Method of detecting a biological activity

ABSTRACT

The present invention provides method of detecting a predetermined biological activity. The method includes using an aqueous mixture comprising a first indicator reagent with a first absorption spectrum and a second indicator reagent. The second indicator reagent is converted by the predetermined biological activity to a second biological derivative with a second emission spectrum. The first absorbance spectrum includes detectable absorbance in at least a portion of wavelengths present in the second emission spectrum. The first indicator reagent is received and concentrated from an aqueous liquid by a substrate, facilitating the detection of the second biological derivative.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/825,881, filed Mar. 25, 2013 (now U.S. Pat. No. 8,802,392), which isa national stage filing under 35 U.S.C. 371 of PCT/US2011/058212, filed28 Oct. 2011, which claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 61/408,966 and 61/408,977, both filed on Nov. 1,2010, which are incorporated herein by reference in their entirety.

BACKGROUND

Methods for the detection of a cell (e.g., a pathogenic microorganism ora cancer cell) in a sample often involve the detection of a biologicalactivity (e.g., an enzyme activity or a biochemical pathway) known to beassociated with the particular cell. Often, the biological activity isdetected using an indicator system that is changed via the biologicalactivity to a biological derivative.

Some methods employ two indicator systems to detect a particular type ofcell. For example, methods to detect E. coli can include a firstindicator system that includes lactose in combination with a pHindicator. The fermentation of lactose to organic acids indicates thepresence of a member of the coliform bacteria (which includes E. coliand other enteric microorganisms). The methods also include a secondindicator system, such as 4-methylumbelliferyl-β-D-glucuronic acid,which is used to detect the enzyme β-glucuronidase, an enzyme found inmost E. coli. Thus, in a method employing both indicator systems, theaccumulation of acidic end products from lactose, along with theaccumulation of a fluorescent compound (4-methylumbelliferone) canindicate the presence of E. coli in a sample.

The detection of a particular biological activity in a sample may beindicative of viable cells in the sample. Bacterial spores, for example,include biological activities (e.g., enzyme activities such asα-glucopyranosidase or β-glucopyranosidase) that may be used in methods(e.g., including rapid methods) detect the presence of viable spores ina sample. Destruction of one of these or other biological activities canbe used to verify and/or validate the efficacy of a sterilizationprocess.

SUMMARY OF THE INVENTION

The present disclosure generally relates to methods to detect abiological activity in a sample. The inventive methods provide means todetect biological activity with at least two (e.g., “first” and“second”) indicator reagents. The methods provide for rapid, sensitivedetection of a biological derivative of the second indicator reagent ina reaction mixture that, initially, includes a high enough concentrationof a first indicator reagent to interfere with the detection of thebiological derivative.

In one aspect, the present disclosure provides a method of detecting abiological activity. The method can comprise providing a sample that maycomprise a source of one or more predetermined biological activities, afirst indicator system comprising a first indicator reagent with a firstabsorbance spectrum, a second indicator system comprising a secondindicator reagent that is converted by a second predetermined biologicalactivity to a second biological derivative with a second emissionspectrum, and a substrate that receives and concentrates the firstindicator reagent from an aqueous mixture. The first indicator reagentcan be converted by a first predetermined biological activity to a firstbiological derivative. The first absorbance spectrum can includedetectable absorbance in at least a portion of wavelengths present inthe second emission spectrum. The method further can comprise forming afirst aqueous mixture comprising the sample, the first indicatorreagent, and the second indicator reagent. The method further cancomprise bringing the first aqueous mixture into fluid communicationwith the substrate to form a second aqueous mixture in which theconcentration of the first indicator reagent is lower than theconcentration of the first indicator reagent in the first aqueousmixture. The method further can comprise detecting a presence or absenceof fluorescence from the second biological derivative.

In some embodiments, detecting the presence or absence of fluorescencefrom the second biological derivative can comprise detecting thepresence or absence of fluorescence in the second aqueous mixture. Insome embodiments, the method further can comprise observing thesubstrate to detect the first indicator reagent or the first biologicalderivative. In any of the above embodiments, a concentration of firstindicator reagent in the first aqueous mixture can be sufficient toprevent the detection of an otherwise detectable amount of the secondbiological derivative. In any of the above embodiments, the methodfurther can comprise providing a nutrient to facilitate growth of abiological cell, wherein forming the first aqueous mixture comprisesforming a mixture that includes the nutrient. In any of the aboveembodiments, the method further can comprise exposing the biologicalactivity to a sterilant. The sterilant can be selected from the groupconsisting of steam, ethylene oxide, hydrogen peroxide, formaldehyde,and ozone.

In any of the above embodiments, the first indicator reagent cancomprise a chromophore, wherein detecting a biological derivative of thefirst reagent comprises detecting a color. In any of the aboveembodiments, the first indicator reagent can comprise a chromogenicindicator. In any of the above embodiments, the first indicator reagentcan comprise a pH indicator or an enzyme substrate. In some embodiments,the first indicator reagent can comprise bromocresol purple.

In any of the above embodiments, the second indicator reagent cancomprise a fluorogenic compound. The fluorogenic compound can comprise afluorogenic enzyme substrate.

In any of the above embodiments, detecting the presence or absence ofthe second biological derivative further can comprise measuring aquantity of the second biological derivative. In any of the aboveembodiments, detecting the presence or absence of the first biologicalderivative further can comprise measuring a quantity of the firstbiological derivative.

In any of the above embodiments, the method further can compriseproviding an instrument that detects the first indicator reagent or thebiological derivative of the second indicator reagent and using theinstrument to detect the first indicator reagent or the biologicalderivative of the second indicator reagent.

In some embodiments, the method further can comprise providing aninstrument that detects the first indicator reagent or the secondbiological derivative and using the instrument to detect the firstindicator reagent or the second biological derivative. In someembodiments, the method further can comprise providing an instrumentthat detects the first indicator reagent and the second biologicalderivative and using the instrument to detect the first indicatorreagent and the second biological derivative.

In another aspect, the present disclosure provides a method of detectinga biological activity. The method can comprise providing a housing, acontainer, a source of a second predetermined biological activity, and asubstrate. The housing can comprise first and second chambers. Thecontainer can contain a first aqueous liquid. The container can bedisposed in the first chamber. At least a portion of the container canbe frangible. The first aqueous liquid can comprise a first indicatorsystem comprising a first indicator reagent with a first absorbancespectrum and a second indicator system comprising a second indicatorreagent that is converted by a predetermined biological activity to asecond biological derivative with a second emission spectrum, whereinthe first absorbance spectrum includes detectable absorbance in at leasta portion of wavelengths present in the second emission spectrum. Thefirst indicator reagent can be converted by a first predeterminedbiological activity to a first biological derivative. The source of thepredetermined biological activity can be disposed in the second chamber.The substrate can be disposed in the housing and can receive andconcentrate the first indicator reagent from the first aqueous liquid.The method further can comprise bringing the first aqueous mixture intofluid communication with the substrate to form a second aqueous mixturein which the concentration of the first indicator reagent is lower thanthe concentration of the first indicator reagent in the first aqueousmixture. The method further can comprise detecting a presence or absenceof fluorescence from the second biological derivative. In someembodiments, detecting the presence or absence of fluorescence from thesecond biological derivative can comprise detecting the presence orabsence of fluorescence in the second aqueous mixture. In someembodiments, bringing the first aqueous mixture into fluid communicationwith the substrate to form a second aqueous liquid can comprisefracturing at least a portion of the frangible container. In someembodiments, the biological sterilization indicator further cancomprises a breaker disposed in the housing, wherein fracturing thefrangible container comprises urging the container and the breakeragainst one another. In some embodiments, the housing of the biologicalsterilization indicator can include a first portion and a secondportion. The second portion can be adapted to be coupled to the firstportion, the second portion being movable with respect to the firstportion, when coupled to the first portion, between a first position anda second position. The method further can comprise moving the secondportion of the housing from the first position to the second position.

In another aspect, the present disclosure provides a system to detect apredetermined biological activity. The system can comprise a firstindicator system comprising a first indicator reagent with a firstabsorbance spectrum, a second indicator system comprising a secondindicator reagent that is converted by a predetermined biologicalactivity to a second biological derivative with a second emissionspectrum, a vessel configured to hold a liquid medium, a substrate thatreceives and concentrates the first indicator reagent from an aqueousmixture, and an instrument configured to receive the vessel and todetect the first indicator reagent or a biological derivative of thesecond indicator reagent. The first indicator reagent can be convertedby a first predetermined biological activity to a first biologicalderivative. The first absorbance spectrum includes detectable absorbancein at least a portion of wavelengths present in the second emissionspectrum. In some embodiments, the instrument can be configured todetect the first biological derivative. In some embodiments, the systemfurther can comprise a processor. In any of the above embodiments of thesystem, the instrument further can be configured to regulate atemperature of a liquid medium. In any of the above embodiments of thesystem, the instrument can be configured to detect both the firstindicator reagent and the second biological derivative.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a substrate can be interpretedto mean “one or more” substrates.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

“Biological activity”, as used herein, refers to any specific catalyticprocess or groups of processes associated with a biological cell.Nonlimiting examples of biological activities include catabolic enzymeactivities (e.g., carbohydrate fermentation pathways), anabolic enzymeactivities (e.g. synthetic pathways for nucleic acids, amino acids, orproteins), coupled reactions (e.g., a metabolic pathway)biomolecule-mediated redox reactions (e.g., electron transport systems),and bioluminescent reactions. “Predetermined” biological activity meansthat the method is directed toward the detection of a specificbiological process (e.g., an enzyme reaction) or group of biologicalprocesses (e.g., a biochemical pathway). It will be appreciated by aperson having ordinary skill in the art that certain predeterminedbiological activities may be associated with a particular type of cell(e.g., a cancer cell or a microorganism) or a pathological process.

“Biological derivative”, as used herein, refers to a product abiological activity. This includes, for example, products of enzymereactions and biological electron transport systems.

“Biomolecules”, as used herein, can be any chemical compound that occursnaturally in living organisms, as well as derivatives or fragments ofsuch naturally occurring compounds. Biomolecules consist primarily ofcarbon and hydrogen, along with nitrogen, oxygen, phosphorus, andsulfur. Other elements sometimes are incorporated but are much lesscommon. Biomolecules include, but are not limited to, proteins,polypeptides, carbohydrates, polysaccharides, lipids, fatty acids,steroids, prostaglandins, prostacylins, vitamins, cofactors, cytokines,and nucleic acids (including DNA, RNA, nucleosides, nucleotides,purines, and pyrimidines), metabolic products that are produced byliving organisms including, for example, antibiotics and toxins.Biomolecules may also include derivatives of naturally occurringbiomolecules, such as a protein or antibody that has been modified withchemicals (e.g., oxidized with sodium periodate). Biomolecules may alsoinclude crosslinked naturally occurring biomolecules, or a crosslinkedproduct of a naturally occurring biomolecule with a chemical substance.Thus, “biomolecule” includes, but is not limited to, both unmodified andmodified molecules (e.g., glycosylated proteins, oxidized antibodies)and fragments thereof (e.g., protein fragments). Fragments ofbiomolecules can include those resulting from hydrolysis due tochemical, enzymatic, or irradiation treatments, for example.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a top perspective view of a substrate and a vessel holding aliquid medium comprising an indicator reagent.

FIG. 1B is a top perspective view of the vessel of FIG. 1A immediatelyafter immersion of the substrate of FIG. 1A into the liquid medium.

FIG. 1C is a top perspective view of the vessel of FIG. 2A after aperiod of time.

FIG. 2 is a drawing of a u.v.-visible absorbance spectrum of an aqueoussolution of bromocresol purple and a fluorescence emission spectrum of asolution of 4-methylumbelliferone.

FIG. 3 is a block diagram of one embodiment of a method of detecting abiological activity according to the present disclosure.

FIG. 4 is a front perspective view of a biological sterilizationindicator according to one embodiment of the present disclosure, thebiological sterilization indicator including a housing that includes afirst portion and a second portion.

FIG. 5 is a rear perspective view of the biological sterilizationindicator of FIG. 4.

FIG. 6 is a front exploded view of the biological sterilizationindicator of FIGS. 4-5.

FIG. 7 is a side cross-sectional view of the biological sterilizationindicator of FIGS. 4-6, taken along line 4-4 of FIG. 4, the biologicalsterilization indicator shown in a first state, and the second portionof the housing of the biological sterilization indicator shown in afirst position.

FIG. 8 is a top cross-sectional view of the biological sterilizationindicator of FIGS. 4-6, taken along line 5-5 of FIG. 6.

FIG. 9 is a side cross-sectional view of the biological sterilizationindicator of FIGS. 4-8, the biological sterilization indicator shown ina second state, and the second portion of the housing of the biologicalsterilization indicator shown in a second position.

FIG. 10 is a top cross-sectional view of the biological sterilizationindicator of FIGS. 4-9, with portions removed for clarity.

DETAILED DESCRIPTION

The present disclosure relates to a rapid method for detecting abiological activity. The method includes the use of two or moreindicator reagents. The method includes providing a liquid mixturecomprising a first and a second indicator reagent, wherein the firstindicator reagent is present in the mixture at a concentrationsufficient to interfere with the detection (e.g., optical detection) ofan otherwise detectable quantity of a biological derivative of thesecond indicator reagent. The inventive method provides rapid, sensitivedetection of a biological activity by sequestering at least a portion ofthe interfering quantity of first indicator reagent from the bulk of theliquid mixture in order to facilitate detection of the biologicalderivative of the second indicator reagent. The method further providesa means to more easily observe the first indicator reagent or abiological derivative thereof. The inventive method can be used in asystem for the automated detection of a biological activity.

The inventive system and/or method of the present disclosure can be usedto detect a biological activity (e.g., an activity associated with anenzyme, a cell, or a microorganism). In some embodiments, the inventivesystem and/or method can be used, for example, to detect a biologicalactivity associated with a particular type of microorganism (e.g., avegetative cell or a spore) that has survived exposure to a process(e.g., a disinfection process, a food or beverage preparation process, asterilization process).

The inventive method relates to the detection of a biological activityin a sample. The sample can be any sample that includes a biologicalactivity as defined herein. Nonlimiting examples of suitable samplesinclude suspensions or cultures of cells (e.g., mammalian cells, insectcells, yeast cells, filamentous fungi, bacterial cells), environmentalsamples (e.g., surface swabs), food (e.g., raw materials, in-processsamples, and finished-product samples), beverages, clinical samples(e.g., blood, urine, sputum, tissue, mucous, feces, wound exudate, pus),and water (e.g., surface water, potable water, process water).

Microorganisms (e.g., bacteria, fungi, viruses) are a source ofbiological activity and can be analyzed in a test sample that may bederived from any source, such as a physiological fluid, e.g., blood,saliva, ocular lens fluid, synovial fluid, cerebral spinal fluid, pus,sweat, exudate, urine, mucus, lactation milk, or the like. Further, thetest sample may be derived from a body site, e.g., wound, skin, nares,scalp, nails, etc.

Samples of particular interest include mucus-containing samples, such asnasal samples (from, e.g., anterial nares, nasopharyngeal cavity, nasalcavities, anterior nasal vestibule, etc.), as well as samples from theouter ear, middle ear, mouth, rectum, vagina, or other similar tissue.Examples of specific mucosal tissues include buccal, gingival, nasal,ocular, tracheal, bronchial, gastrointestinal, rectal, urethral,ureteral, vaginal, cervical, and uterine mucosal membranes.

Besides physiological fluids, other test samples may include otherliquids as well as solid(s) dissolved in a liquid medium. Samples ofinterest may include process streams, water, soil, plants or othervegetation, air, surfaces (e.g., contaminated surfaces), and the like.Samples can also include cultured cells. Samples can also includesamples on or in a device comprising cells, spores, or enzymes (e.g., abiological indicator device).

Solid samples may be disintegrated (e.g., by blending, sonication,homogenization) and may be suspended in a liquid (e.g., water, buffer,broth). In some embodiments, a sample-collection device (e.g., a swab, asponge) containing sample material may be used in the method.Alternatively, the sample material may be eluted (e.g., rinsed, scraped,expressed) from the sample-collection device before using the samplematerial in the method. In some embodiments, liquid or solid samples maybe diluted in a liquid (e.g., water, buffer, broth).

Suitable samples also liquid and/or solid samples that have been exposedto a sterilant. Nonlimiting examples of these samples include sporesuspensions, spore strips, and coupons of various materials onto which asuspension of spores or vegetative microbial cells have been applied.

Suitable samples also include cell-suspension media (e.g., culturebroth, semi-solid cell culture media, and tissue culture media,filtrate) that contain cells or previously contained cells. Suitablesamples also include cell lysates. Cell lysates may be produced bychemical means (e.g., detergents, enzymes), mechanical means (sonicvibration, homogenization, French Press), or by other cell lytic meansknown in the art.

FIGS. 1A through 1C illustrate the process of receiving andconcentrating from a liquid medium an indicator reagent (or a biologicalderivative thereof) onto or into a substrate according to the presentdisclosure. FIG. 1A shows a top perspective view of one embodiment of asubstrate 30 and a vessel 10 containing a liquid mixture 20 comprising acolored indicator reagent. FIG. 1B shows a top perspective view of thevessel 10 of FIG. 1A immediately after immersing the substrate 30 in theliquid mixture 20. FIG. 1C shows a top perspective view of the vessel 10of FIG. 1B after a period of time sufficient to permit the substrate 30to receive and concentrate the colored indicator reagent from the liquidmixture 20. It can be seen in FIG. 1C that the color of the liquidmixture has become less intense, while the substrate 30 has received andretained the colored indicator reagent and, thereby, has changed fromits initial colorless state to a colored state.

In some embodiments, the substrate may passively receive and concentratethe indicator reagent or biological derivative thereof (e.g., by simplediffusion of the reagent or derivative through the liquid medium).Alternatively or additionally (not shown), the substrate may activelyreceive and concentrate the indicator reagent and/or biologicalderivative (e.g., the substrate may be moved relative to the liquid viamixing or tumbling and/or the liquid medium may be moved relative to thesubstrate via fluid flow that is generally lateral, tangential, ororthogonal to a major surface of the substrate).

Indicator Reagents

The prior art includes a number of chromic and fluorogenic enzymesubstrates of diverse origin which are known, commercially available,that have been used in methods to detect predetermined biologicalactivities, and are suitable for use as the first or second indicatorreagent according to the present disclosure. Among these are a varietyof fluorogenic 4-methylumbelliferyl derivatives (hydrolysable to4-methylumbelliferone); derivatives of 7-amido-4-methyl-coumarin, e.g.as disclosed in GB Patent No. 1,547,747 and European Patent No.0,000,063, each of which is incorporated herein by reference in itsentirety; diacetylfluorescein derivatives; and fluorescamine.

The first indicator reagent, according to the present disclosure,comprises a reagent that has a first absorption spectrum and, thus, itabsorbs light in the ultraviolet and/or visible wavelengths of theelectromagnetic spectrum.

In some embodiments, the first indicator reagent can be an indicator dye(e.g., a pH indicator dye, a redox dye). The specific indicator dye usedto detect any given biological activity will be selected according tocriteria that are known in the art, including, for example,compatibility (e.g., preferably non-inhibitory) with the biologicalactivity to be detected, solubility, detection system (e.g., visualand/or automated).

In any of the embodiments of the method, the indicator dye may be a pHindicator suitable to detect the biological activity. The indicator dyecan be selected according to criteria known in the art such as, forexample, pH range, compatibility with the biological activity, andsolubility. In some embodiments, a salt form of the pH indicator may beused, for example, to increase the solubility of the pH indicator in anaqueous mixture. Nonlimiting examples of suitable pH indicator dyesinclude, for example, thymol blue, tropeolin OO, methyl yellow, methylorange, bromophenol blue, bromocresol green, methyl red, bromothymolblue, phenol red, neutral red, phenolphthalein, thymolphthalein,alizarin yellow, tropeolin O, nitramine, trinitrobenzoic acid, thymolblue, bromophenol blue, tetrabromophenol blue, bromocresol green,bromocresol purple, methyl red, bromothymol blue, phenol red, Congo red,and cresol red.

In any of the embodiments of the method, the indicator dye may be anoxidation-reduction indicator (also called a redox indicator) suitableto detect the biological activity. Oxidation-reduction indicator dyesmay be pH-dependent or pH-independent. Nonlimiting examples ofoxidation-reduction indicator dyes include 2,2′-Bipyridine (Ru complex),Nitrophenanthroline (Fe complex), N-Phenylanthranilic acid,1,10-Phenanthroline (Fe complex), N-Ethoxychrysoidine, 2,2′-Bipyridine(Fe complex), 5,6-Dimethylphenanthroline (Fe complex), o-Dianisidine,Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine,Viologen, Sodium 2,6-Dibromophenol-indophenol, Sodium2,6-Dichlorophenol-indophenol, Sodium o-Cresol indophenol, Thionine(syn. Lauth's violet), Methylene blue, Indigotetrasulfonic acid,Indigotrisulfonic acid, Indigodisulfonic acid, Indigomonosulfonic acid,Phenosafranin, Safranin T, and Neutral red.

In some embodiments, the first indicator reagent can be asulfonphthalein pH indicator (e.g. bromocresol purple), as shown inExample 4. The sulfonphthalein pH indicator (e.g., bromocresol purple)can be present in the aqueous mixture at a concentration of about 0.03 gper liter. The sulfonphthalein pH indicator can be received andconcentrated by a substrate (e.g. a charged nylon substrate such as, forexample, MAGNAPROBE 0.45 micron charged nylon membrane, part numberNP0HY00010, available from GE Osmonics Labstore, Minnetonka, Minn.). Thesubstrate can be configured as a generally planar strip (e.g. a stripthat is about 3 mm by about 10 mm).

The second indicator reagent, according to the present disclosure, canbe converted to a second biological derivative. The second biologicalderivative comprises a reagent that has a second absorption spectrum.Furthermore, the second biological derivative has a characteristicsecond emission spectrum (e.g., a fluorescent emission spectrum). Insome embodiments, the second biological derivative has a characteristicsecond absorption spectrum that includes wavelengths in the ultravioletportion of the electromagnetic energy spectrum. The second emissionspectrum of the second biological derivative may include wavelengths inthe visible portion of the electromagnetic energy spectrum.

Suitable compounds for use as a second indicator reagent includefluorogenic compounds (e.g., fluorogenic enzyme substrates). Fluorogenicenzyme substrates include 4-methylumbelliferyl derivatives,7-amido-4-methylcoumarin derivatives, and diacetylfluoresceinderivatives.

Suitable 4-methylumbelliferyl derivatives include, for example:4-methylumbelliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside;4-methylumbelliferyl acetate;4-methylumbelliferyl-N-acetyl-β-D-galactosaminide;4-methylumbelliferyl-N-acetyl-α-D-glucosaminide;4-methylumbelliferyl-N-acetyl-β-D-glucosaminide;2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid;4-methylumbelliferyl α-L-arabinofuranoside; 4-methylumbelliferylα-L-arabinoside; 4-methylumbelliferyl butyrate; 4-methylumbelliferylβ-D-cellobioside; methylumbelliferyl β-D-N,N′ diacetyl chitobioside;4-methylumbelliferyl elaidate; 4-methylumbelliferyl β-D-fucoside;4-methylumbelliferyl α-L-fucoside; 4-methylumbelliferyl β-L-fucoside;4-methylumbelliferyl α-D-galactoside; 4-methylumbelliferylβ-D-galactoside; 4-methylumbelliferyl α-D-glucoside;4-methylumbelliferyl β-D-glucoside; 4-methylumbelliferylβ-D-glucuronide; 4-methylumbelliferyl p-guanidinobenzoate;4-methylumbelliferyl heptanoate; 4-methylumbelliferylα-D-mannopyranoside; 4-methylumbelliferyl β-D-mannopyranoside;4-methylumbelliferyl oleate; 4-methylumbelliferyl palmitate;4-methylumbelliferyl phosphate; 4-methylumbelliferyl propionate;4-methylumbelliferyl stearate; 4-methylumbelliferyl sulfate;4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotriose;4-methylumbelliferyl 2,3,5-tri-o-benzoyl-α-L-arabinofuranoside;4-methylumbelliferyl-p-trimethylammonium cinnamate chloride; and4-methylumbelliferyl β-D-xyloside.

Suitable 7-amido-4-methylcoumarin derivatives include, for example:L-alanine-7-amido-4-methylcoumarin; L-proline 7-amido-4-methylcoumarin;L-tyrosine-7-amido-4-methylcoumarin; L-leucine-7-amido-4-methylcoumarin;L-phenylalanine-7-amido-4-methylcoumarin; and7-glutarylphenylalanine-7-amido-4-methylcoumarin.

Suitable peptide derivatives of 7-amido-4-methyl coumarin include, forexample: N-t-BOC-Ile-Glu-Gly-Arg 7-amido-4-methylcoumarin;N-t-BOC-Leu-Ser-Thr-Arg 7-amido-4-methylcoumarin; N-CBZ-Phe-Arg7-amido-4-methyl-coumarin; Pro-Phe-Arg 7-amido-4-methylcoumarin;N-t-BOC-Val-Pro-Arg 7-amido-4-methylcoumarin; and N-glutaryl-Gly-Arg7-amido-4-methylcoumarin.

Suitable diacetylfluorescein derivatives include, for example,fluorescein diacetate, fluorescein di-(β-D-galactopyranoside), andfluorescein dilaurate.

Where the biological activity to be detected is alpha-D-glucosidase,chymotrypsin, or fatty acid esterase, e.g., from Geobacillusstearothermophilus, preferred fluorogenic enzyme substrates are4-methylumbelliferyl-alpha-D-glucoside,7-glutarylphenylalanine-7-amido-4-methylcoumarin, or4-methylumbelliferyl heptanoate, respectively. Where the biologicalactivity to be detected is alpha-L-arabinofuranosidase, e.g., derivedfrom Bacillus subtilis, a preferred fluorogenic enzyme substrate is4-methylumbelliferyl-alpha-L-arabinofuranoside. Where the biologicalactivity to be detected is beta-D-glucosidase, e.g., derived fromBacillus subtilis, a preferred fluorogenic enzyme substrate is4-methylumbelliferyl-beta-D-glucoside.

In order to carry out the method of the present invention in detecting abiological activity comprising an enzyme, the operator should beknowledgeable concerning the enzyme activity to be detected and theenzyme substrates that will react with the enzyme so as to produce aproduct which can be detected either by its fluorescence, color, etc.(see M. Roth, Methods of Biochemical Analysis, Vol. 7, D. Glock, Ed.,Interscience Publishers, New York, N.Y., 1969, which is incorporatedherein by reference in its entirety). The appropriate enzyme substrateto be utilized will depend upon the biological activity to be detected.

Methods of the present disclosure include a first indicator reagent witha first absorption spectrum and a second indicator reagent that isconverted by a biological activity to a second biological derivativewith a second emission spectrum, wherein the first absorption spectrumat least partially overlaps the second emission spectrum. Thus, whenboth the first indicator reagent and the second biological derivativeare present in a liquid mixture, the first indicator reagent may absorbat least a portion of the light emitted by the second indicator reagent,thereby diminishing the ability to detect the second biologicalderivative.

A drawing can illustrate the relationship between a first indicatorreagent and a second biological derivative according to the presentdisclosure. FIG. 2 shows the absorbance spectrum of Bromocresol Purple(hereinafter, called “BCP”), an exemplary first indicator reagent, andthe fluorescence emission spectrum of 4-methylumbelliferone(hereinafter, called “4MU”), a possible biological derivative of4-methylumbelliferyl β-D-glucoside, an exemplary second indicatorreagent. The spectra were obtained as described in Examples 1 and 2.

Line “A”, which shows the absorbance spectrum of BCP, indicates anabsorbance maximum in the visible range around 600 nm, with relativelyless absorbance by BCP in the 425-550 nm wavelengths. The data show anabsorbance peak in the visible wavelengths around 600 nm and anabsorbance peak in the ultraviolet wavelengths at <330 nm. Line “B”,which shows the fluorescence emission spectrum of 4MU indicates anemission maximum around 450 nm, with relatively less emission in theranges from 375-425 nm and from 475-525 nm. It can be seen in FIG. 2that the absorbance spectrum of BCP substantially overlaps the entirefluorescence emission peak (centered around 450 nm) of 4MU.

A person of ordinary skill in the relevant art will recognize that theamount of absorbance of any particular wavelength of light by a solutioncontaining a first indicator reagent will be influenced by theconcentration of first indicator reagent in the solution and the molarextinction coefficient of the indicator reagent at the selectedwavelength. The skilled person will also recognize that the amount oflight emission of any particular wavelength by a solution containing abiological derivative of a second indicator reagent will be influencedby the concentration of the second biological derivative in the solutionand the fluorescence quantum yield of the biological derivative.Therefore, the concentration of the first indicator reagent in theliquid mixture can be selected in conjunction with an appropriatesubstrate to permit i) the substrate to remove enough first indicatorsubstrate from the liquid mixture to allow more sensitive detection ofthe second biological derivative and ii) the first indicator reagent (orbiological derivative thereof) to be easily detected on the substratematerial.

The combination of bromocresol purple and4-methylumbelliferyl-α-D-glucoside represents an example of suitablefirst and second indicator reagents, respectively, according to thepresent disclosure. This combination can be used to detect a firstbiological activity such as the fermentation of a carbohydrate to acidend products and a second biological activity such as -α-D-glucosidaseenzyme activity, for example. These activities can indicate the presenceor absence of a viable spore following the exposure of a biologicalsterilization indicator to a sterilization process, for example. Thebromocresol purple can be used at a concentration of about 0.03 g/L inthe aqueous mixture, for example. The 4-methylumbelliferyl-α-D-glucosidecan be used, for example, at a concentration of about 0.05 to about 0.5g/L (e.g., about 0.05 g/L, about 0.06 g/L, about 0.07 g/L, about 0.08g/L, about 0.09 g/L, about 0.1 g/L, about 0.15 g/L, about 0.2 g/L, about0.25 g/L, about 0.3 g/L, about 0.35 g/L, about 0.4 g/L, about 0.45 g/L,about 0.5 g/L). in the aqueous mixture.

Thus, according to the present disclosure, the first indicator reagentmay interfere with the detection of an otherwise detectable amount ofthe biological derivative of the second indicator reagent. The spectralinterference between any proposed first and second indicator reagentscan be demonstrated by a person of ordinary skill in the art byperforming the following simple experiment.

First, the operator makes a relatively-dilute, butfluorescently-detectable, aqueous solution of the expected biologicalderivative of the proposed second indicator reagent. For example, if thesecond indicator reagent is a 4-methylumbelliferyl compound, theexpected biological derivative is 4MU. The solution can contain, forexample, about 0.05 to 0.2 micrograms per milliliter 4MU. Next, theoperator adds an effective amount of the proposed first indicatorreagent. For example, if BCP is the proposed first indicator reagent, itcan be added at a concentration (e.g., 0.04 milligrams per milliliter)that is used in microbiological growth media for the detection offermentative microorganisms. By comparing the fluorescence of the 4MUsolutions with and without the BCP, it can be determined whether thefirst indicator reagent (in this example, the BCP) can interfere withthe detection of the biological derivative of the second indicatorreagent (in this case, the 4MU). The operator can then test whetheradding reduced amounts of BCP to the 4MU solution improves the detectionof relatively low concentrations of 4MU. This type of experiment easilycan be performed with any combination of first and second indicatorreagents. An example of this procedure is shown in Example 3.

Substrate

Suitable substrates, according to the present disclosure, are configuredto receive and concentrate the indicator reagent. The ability of thesubstrate to concentrate the indicator reagent or biological derivativethereof can be affected by one or more of a variety of forces known inthe art and discussed herein. Thus, a person of ordinary skill in theart may select a substrate that is known to be positively-charged toconcentrate an indicator reagent (or biological derivative thereof) thatis known to be negatively-charged, for example. Conversely, a person ofordinary skill in the art may select a substrate that is known to benegatively-charged to concentrate an indicator reagent (or biologicalderivative thereof) that is known to be positively-charged. A person ofordinary skill in the art may select a substrate that is known to havehydrophobic properties to concentrate an indicator reagent (orbiological derivative thereof) that is known to comprise hydrophobicportions that would be retained by a hydrophobic substrate.Additionally, a person of ordinary skill in the art may easily select asuitable substrate material by contacting, for a period of time, acandidate substrate material with a liquid comprising the indicatorreagent or biological derivative thereof and analyzing the substrate todetermine whether a detectable amount of the indicator reagent orderivative thereof accumulates onto or in the substrate.

It will be apparent to a person of ordinary skill in the art that thesubstrate material can be selected according to known properties of theindicator reagent or the biological derivative thereof. For example, apositively-charged substrate may be selected for use in the method whenthe biological derivative of the indicator reagent is anegatively-charged molecule. Furthermore, a negatively-charged substratemay be selected for use in the method when the biological derivative ofthe indicator reagent is a positively-charged molecule.

Alternatively, the suitability of any given substrate material for usewith a given first indicator reagent in the inventive method can bereadily determined using the following experimental approach. In asuitable vessel (e.g., a test tube), a source of predeterminedbiological activity (e.g., microbial cells capable of fermenting acarbohydrate to acidic end products) can be added with a first indicatorreagent (e.g., a pH indicator) to a liquid medium selected to facilitatethe biological activity (e.g. a broth medium comprising the fermentablecarbohydrate). The liquid medium can be contacted with a candidatesubstrate under conditions to facilitate the predetermined biologicalactivity and the substrate can be removed from the medium, optionallyrinsed and/or blotted to remove excess liquid, and observed visually orinstrumentally (e.g., with a spectroreflectometer or a fluorometer) todetermine whether the substrate material concentrated the firstindicator reagent and/or a biological derivative thereof during contactwith the liquid medium. In the illustrative example, a suitablesubstrate/indicator combination would show evidence that either thefirst indicator reagent or the biological derivative thereofconcentrated onto or into the substrate material (out of the liquidmedium) during the contact period. A control reaction without substratematerial can be run to confirm the presence of the biological activityin the mixture.

The substrate may be fabricated in a generally planar sheet form (e.g.,a membrane strip, as shown in FIG. 1A). The size and/or effectivesurface area of the substrate can also affect the ability of thesubstrate to concentrate the indicator reagent (or biological derivativethereof). Preferred materials for the substrate include porous materials(e.g., woven materials, nonwoven materials, a porous membranes,microporous membranes, filter paper). In some embodiments, particularlypreferred substrate materials include charged membranes such as, forexample, charged nylon membranes (e.g., MAGNAPROBE 0.45 micron chargednylon membrane, part number NP0HY00010, available from GE OsmonicsLabstore, Minnetonka, Minn.). Substrates used in the present disclosurecan be fabricated from a variety of materials. U.S. Pat. No. 6,562,297,which is incorporated herein by reference in its entirety, describesmembranes for the immobilization of pH indicators. Nonlimiting examplesof suitable substrate materials include, for example, natural materials(e.g., cellulose), synthetic materials (e.g. nylon), and combinationsand/or derivatives thereof.

Method of Detecting a Biological Activity:

FIG. 3 shows a block diagram of one embodiment of a method to detect oneof a plurality of biological activities according to the presentdisclosure.

The method includes the step 40 of providing a sample that may includeone of a plurality of predetermined biological activities, first andsecond indicator reagents, and a substrate that receives andconcentrates from an aqueous medium the first indicator reagent and,optionally, a biological derivative thereof.

In some embodiments, the method may include the optional step 45 ofexposing the biological activity to a disinfectant, an antibiotic, or asterilant. This optional step may be included to determine the efficacyof a sterilization process or to detect a predetermined biologicalactivity (or microorganism) subsequent to a selective enrichment cultureprocess. Exposing the biological activity to a sterilant may compriseexposing the biological activity to a sterilization process.Sterilization processes include exposing the sample, for example, tosterilants such as steam, dry heat, ethylene oxide, formaldehyde,peroxides, hydrogen peroxide, peracetic acid, ozone, or mixtures thereof(e.g., a mixture of ozone and hydrogen peroxide.

The method includes the step 50 of forming a first aqueous mixturecomprising the sample and the first and second indicator reagents. Thefirst aqueous mixture is formed in an aqueous medium. The source ofbiological activity in the method can be any sample comprising, orsuspected of comprising one or more biological activities, as describedherein. “Aqueous medium”, as used herein, refers to an aqueous liquid inwhich the first and second indicator reagents are or can be dissolved orsuspended. Preferably, the medium does not substantially interfere withthe detection of a predetermined biological activity to be detected. Insome embodiments, the aqueous medium may comprise a component (i.e., abuffering agent) to adjust the pH of the medium. The aqueous mediumfurther may comprise a reagent (e.g., a detergent, a cofactor, a celllysis agent) that is known in the art to facilitate the detection of oneor more biological activities.

In some embodiments, the sample comprises water and, thus, the sampleitself may be considered an aqueous medium. In any embodiment, thesample may optionally be mixed with a second liquid (e.g., an aqueousmedium, a diluent, a buffer, a solution to neutralize a disinfectant)before mixing the sample with the first and second indicator reagents.

In some embodiments, the aqueous medium can be combined with the firstand/or second indicator reagents before the medium is mixed with thesample. In some embodiments, the first and second indicator reagents andthe sample can be added sequentially to the aqueous medium to form thefirst aqueous mixture. In some embodiments, the first and secondindicator reagents can be combined with an aqueous medium and the samplesimultaneously to form the first aqueous mixture. In any of theembodiments, either or both of the first and second indicator reagentsinitially may be in the form of a dry reagent, a liquid, a gel, or afilm before the reagent is combined with an aqueous medium and/or asample to form the first aqueous mixture.

The first indicator reagent can be any suitable reagent describedherein. Because the first indicator reagent is selected to detect apredetermined biological activity, the chemical nature of the firstindicator reagent and biological derivatives thereof are known and,thus, suitable substrate materials can be identified as describedherein. The second indicator reagent can be any suitable reagentdescribed herein.

In any embodiment of the method, forming the first aqueous mixture cancomprise forming a first aqueous mixture that includes a nutrient. Thenutrient can be provided to facilitate the growth of a target cell ormicroorganism, for example, and may be provided as a mixture ofnutrients. Nutrients and nutrient media to facilitate the growth ofmicroorganisms are known in the art and can be found, for example, inthe “Handbook of Microbiological Media” by Ronald Atlas, published byCRC Press, Boca Raton, Fla. Matner et al. (U.S. Pat. No. 5,073,488)describes a nutrient medium for the growth and detection of bacterialspores in a biological sterilization indicator. Nutrients and nutrientmedia for facilitating the growth of eukaryotic cells (e.g., mammaliancells, insect cells) are also known in the art and include, for example,sugars (e.g., glucose), amino acids, vitamins (e.g., thiamin, niacin),choline, inositol, serum, and mixtures thereof.

Methods of the present disclosure further include the step 60 ofbringing the first aqueous mixture into fluid communication with thesubstrate to form a second aqueous mixture. Typically, the process ofbringing the first aqueous mixture into fluid communication with thesubstrate occurs in a vessel (e.g., a tube, a bottle, a flask, amicrowell). In any of the embodiments, the vessel may be sealed tominimize evaporation and/or to prevent contamination by an exogenousbiological activity, for example. In any of the embodiments, bringingthe first aqueous mixture into fluid communication with the substratemay include contacting the liquid mixture and the substrate underconditions that facilitate the predetermined biological activity. Aperson of ordinary skill in the art will recognize conditions thatfacilitate the predetermined biological activity. The conditions mayinclude, for example, the pH, ionic strength or buffering capacity ofthe mixture; the concentration of first and/or second indicatorreagents; presence of cofactors in the mixture or vessel; and/ortemperature of the mixture.

In any of the embodiments of the method, bringing the first aqueousmixture into fluid communication with the substrate can includecontrolling the temperature of the mixture. In some embodiments, thetemperature may be controlled at a temperature higher than ambienttemperature (e.g., a temperature that facilitates a reaction, such as acatalytic reaction or binding reaction, involving the biologicalactivity) using a heating block, an incubator, or some other suitableheating means known in the art. In some embodiments, the temperature ofthe mixture may be controlled at a temperature lower than ambienttemperature. In some embodiments, the mixture may be subjected to atransient temperature shift (e.g., a heat shock or a cold shock) tofacilitate the detection of the predetermined biological activity.

Bringing the first aqueous mixture into fluid communication with thesubstrate according to the present disclosure comprises concentratingthe first indicator reagent and; optionally, a biological derivativethereof; onto and/or into the substrate. As a result of this, theconcentration of the first indicator reagent in the second aqueousmixture is lower than the concentration of the first indicator reagentin the first aqueous mixture. As discussed above, the substrate isselected to receive and concentrate the first indicator reagent. Thesubstrate receives the first indicator reagent via contact with theaqueous medium. The substrate retains the first indicator reagent orbiological derivative thereof via a variety of means. Without beingbound by theory, the accumulation of the first indicator reagent orbiological derivative thereof onto and/or into the substrate materialmay occur through one or more of a variety of chemical attractive forcesincluding, but not limited to, ionic interaction, hydrophobicinteraction, van der Waal's forces, and hydrogen bonding, for example.

The process of receiving and concentrating the first indicator reagentor biological derivative thereof by the substrate occurs during theperiod of fluidic communication between the aqueous medium and thesubstrate. During this period of fluidic communication, the firstindicator reagent or biological derivative thereof accumulates on thesubstrate at a rate that may be dependent upon a number of factorsincluding, for example, the concentration of the first indicator reagent(or biological derivative thereof), the surface area of the substratematerial contacting the liquid medium, the porosity of the substrate, acharge density associated with the substrate material, and/or othersubstances in the liquid medium that can interact with the substrateand/or the first indicator reagent (or biological derivative thereof) ina way that interferes with the receiving or concentrating the firstindicator reagent or biological derivative thereof by the substrate.Receiving and concentrating at least a portion of the first indicatorreagent or biological derivative thereof onto the substrate can occurwithin a relatively short contact period (e.g., within several minutes)and may continue over a longer contact period (e.g., up to 1 hour, up to2 hours, up to 4 hours, up to 18 hours, up to 24 hours, up to 7 days, upto two weeks). In some embodiments, the first indicator reagent mayconcentrate onto or into the substrate within a relatively short periodof time (e.g., minutes hours), whereas the first biological derivative,if present, may not be detectably concentrated on or in the substratefor a relatively longer period of time (e.g., hours, days).

During any of the periods of fluidic communication between the aqueousmixture and the substrate described above, the substrate may receive andconcentrate all or a portion of the first indicator reagent (orbiological derivative thereof). In some embodiments, the substratereceives and concentrates at least 5 percent of the first indicatorreagent (or biological derivative thereof). In some embodiments, thesubstrate receives and concentrates at least 10 percent of the firstindicator reagent (or biological derivative thereof). In someembodiments, the substrate receives and concentrates at least 20 percentof the first indicator reagent (or biological derivative thereof). Insome embodiments, the substrate receives and concentrates at least 30percent of the first indicator reagent (or biological derivativethereof). In some embodiments, the substrate receives and concentratesat least 40 percent of the first indicator reagent (or biologicalderivative thereof). In some embodiments, the substrate receives andconcentrates at least 50 percent of the first indicator reagent (orbiological derivative thereof). In some embodiments, the substratereceives and concentrates at least 75 percent of the first indicatorreagent (or biological derivative thereof). In some embodiments, thesubstrate receives and concentrates at least 80 percent of the firstindicator reagent (or biological derivative thereof). In someembodiments, the substrate receives and concentrates at least 90 percentof the first indicator reagent (or biological derivative thereof). Insome embodiments, the substrate receives and concentrates greater than90 percent of the first indicator reagent (or biological derivativethereof). In some embodiments, the substrate receives and concentratesgreater than 95 percent of the first indicator reagent (or biologicalderivative thereof).

Determining that the substrate receives and concentrates the firstindicator reagent (or biological derivative thereof) easily can beaccomplished by bringing a liquid medium comprising the first indicatorreagent (or biological derivative thereof) into fluid communication withthe substrate for a period of time and analyzing the substrate for thepresence of the reagent (or biological derivative thereof), as shown inExample 1. Preferably, any excess liquid medium is removed from thesubstrate (e.g., by blotting or by centrifugation) before analyzing thesubstrate so that the amount of reagent or biological derivativeassociated with the substrate indicates the amount retained by thesubstrate. Suitable analysis methods will be apparent to a person ofordinary skill in the art. For example, a substrate that receives andconcentrates a colored first indicator reagent (e.g., a pH indicator)can be analyzed by reflectance spectroscopy using, for example, anX-Rite model 530P portable Spectrodensitometer.

Thus, when a liquid medium comprising a sample and the first indicatorreagent (or biological derivative thereof) is brought into fluidcommunication with a suitable substrate, the concentration of the firstindicator reagent (or biological derivative thereof) in the bulk liquidmedium decreases as the first indicator reagent (or biologicalderivative thereof) is received and concentrated by the substrate. Thisfeature of the invention facilitates the detection of relatively smallconcentrations of the biological derivative of the second indicatorreagent because at least a portion of the interference (i.e., theabsorption of the fluorescence) by the first indicator reagent isremoved as the first indicator reagent is concentrated onto thesubstrate from the aqueous mixture. In some embodiments, the firstindicator reagent and/or a biological derivative thereof, when in afreely-diffusible form (i.e., in the bulk liquid medium) may inhibit thebiological activity. In these embodiments a further advantage of theinvention is that the substrate can effectively sequester at least aportion of the first indicator reagent, thereby reducing the inhibitionof the biological activity by the first indicator reagent.

Referring back to FIG. 3, the method further may include the optionalstep 65 of facilitating the growth of cells. Facilitating the growth ofcells is used broadly to include providing conditions (e.g., nutrients,germinants, buffers, oxidation-reduction potential, gasses) tofacilitate, for example, the germination of spores, energy metabolism,biosynthesis, and/or cell division. Facilitating the growth of cells mayresult in the amplification of one or more predetermined biologicalactivities from the original sample and, thereby, can improve thesensitivity for detecting the predetermined biological activities.

Methods of the present disclosure further include the step 70 ofdetecting a biological derivative of the second indicator reagent(herein, called “second biological derivative”). In some embodiments,the second biological derivative can be detected in an aqueous medium.Detecting the presence or absence of the second biological is indicativeof the presence or absence, respectively, of the correspondingpredetermined biological activity in the sample.

The second biological derivative can be detected by several means. Insome embodiments, the second biological derivative can be detectedoptically. In some embodiments, detecting the second biologicalderivative may comprise detecting the biological derivative visually. Insome embodiments, detecting the second biological derivative maycomprise detecting the biological derivative using an instrument. Forexample, if the second biological derivative can be detected using anoptical instrument such as a fluorometer.

In any of the embodiments, detecting the presence or absence of thesecond biological derivative thereof may further comprise measuring thequantity of the second biological derivative. Measuring the quantity maybe done by any means known in the art including, for example measuringthe quantity using an instrument (e.g., a fluorometer). In someembodiments, measuring the quantity of second biological derivative maycomprise comparing the fluorescence in the aqueous mixture to afluorescent standard.

In any of the embodiments, methods of the present disclosure optionallyinclude the step 75 of detecting the first indicator reagent or a firstbiological derivative thereof. The means for detecting the firstindicator reagent or the first biological derivative depends upon thenature of the first indicator reagent or the first biologicalderivative, as will be appreciated by a person of ordinary skill in theart. For example, if the first indicator reagent is a chromic (colored)and/or the first biological derivative is a chromic compound, then thefirst indicator reagent and/or the first biological derivative may bedetected optically (either visually or by an instrument (e.g., aspectrophotometer)). In some embodiments, detecting the first indicatorreagent or first biological derivative may further comprise detectingthe first indicator reagent or first biological derivative in a portionof the aqueous mixture that is not associated with the substrate (e.g.,in the bulk liquid). For example, if the first indicator reagent orfirst biological derivative is detected using an optical instrument suchas a spectrophotometer, the optical path does not intersect any portionof the substrate.

In any of the embodiments, detecting the presence or absence of thefirst indicator reagent or first biological derivative may furthercomprise measuring the quantity of the first indicator reagent or firstbiological derivative. Measuring the quantity may be done by any meansknown in the art including, for example measuring the quantity using aninstrument (e.g., a spectrophotometer, a spectrodensitometer).

U.S. Pat. Nos. 5,252,484 and 5,418,167; each of which is incorporatedherein by reference in its entirety; describe an embodiment of a rapidreadout biological indicator wherein the biological indicator comprisesan enzyme carrier (spore strip) and an ampoule contain a solution with4-methylumbelliferyl-α-D-glucoside (“MUG”, a fluorogenic enzymesubstrate) and bromocresol purple (“BCP”, a pH indicator). MUG is knownto be hydrolyzed by enzymatic activity to 4-methylumbelliferone (4MU), afluorescent derivative of MUG. As shown in Examples 1 and 2 of U.S. Pat.No. 5,252,484, the 4MU produced by enzymatic hydrolysis of the MUG canbe detected visually by fluorescence within minutes after the enzymecarrier is brought into fluid communication with the solution containingMUG and BCP.

The present investigators have discovered that the concentration of BCPused in a solution similar to that described in Example 1 of U.S. Pat.No. 5,252,484 is sufficient to interfere with the detection of lowconcentrations of 4MU in an aqueous solution. Removal of at least aportion of the BCP from the solution according to the present disclosurewill permit the detection of smaller quantities of 4MU in a biologicalindicator, thereby permitting earlier detection of biological activity(e.g., spores, enzymes) that have been exposed to a sterilizationprocess and were not thereby inactivated and/or killed.

System for Detecting a Biological Activity

The present disclosure includes a system for detecting a predeterminedbiological activity in a sample. The system can be used according to theinventive method to detect the one or more biological activities in asample. The system includes a first indicator system comprising a firstindicator reagent that can be converted by a first predeterminedbiological activity to a first biological derivative. The firstindicator reagent has a first absorption spectrum and, optionally afirst emission spectrum. The system further includes a second indicatorsystem comprising a second indicator reagent that can be converted by asecond predetermined biological activity to a second biologicalderivative. The second biological derivative has a first absorptionspectrum and a second emission spectrum.

The system further includes an instrument configured to receive a liquidsample that may comprise the first indicator reagent, the secondindicator reagent, the first biological derivative, the secondbiological derivative or any combination of two or more of theforegoing. The instrument may be configured to withdraw the liquidsample from an external container via a “sipper” means, as known in theart of analytical instruments. Alternatively, the instrument may beconfigured to receive a vessel (e.g., a tube, a microwell plate, or thelike) containing the liquid sample.

The instrument is configured to detect the second biological derivative.Optionally, the instrument further can be configured to detect the firstindicator reagent, the second indicator reagent, the first biologicalderivative, or any combination of two or more of the foregoing.

The indicator reagent of the system can be any suitable indicatorreagent, as described herein, to detect the particular predeterminedbiological activity. The first and second indicator reagents may beprovided in a kit, for example, which optionally may include an aqueousmedium (e.g., a buffer, a suspending medium, a diluent) in which to mixthe indicator reagent and the sample. As discussed herein, the samplemay comprise water and, thus, may constitute the aqueous medium.Optionally, the kit may further include a vessel (e.g., a tube, acuvette, or the like) in which to form an aqueous mixture comprising thesample and the first and second indicator reagents. In some embodiments,the system can be used with a biological sterilization indicator suchas, for example, the biological indicators in International PublicationNos. WO 2012/061227 and WO 2012/061226, and the biological indicatorsdescribed in U.S. Pat. No. 5,252,484; each of which is incorporatedherein by reference in its entirety.

Instruments to detect the absorption spectra of chromic compounds areknown in the art and include, for example, a variety ofcommercially-available spectrophotometers and spectrodensitometers.Instruments to detect the emission spectra of fluorescent compounds arealso known in the art and include, for example, a variety ofcommercially-available fluorometers. Such instruments can be readilyadapted to detect an indicator reagent (or biological derivativethereof) associated with a liquid sample and/or a substrate positionedat a predetermined location.

In some embodiments, the substrate can be removed from the aqueousmixture and positioned (e.g., on a surface or in a cuvette) such thatthe indicator reagent (or biological derivative thereof) can be detectedby the instrument. U.S. Pat. No. 6,025,189; which is incorporated hereinby reference in its entirety, describes an instrument configured todetect, at a predetermined location in a self-contained biologicalindicator, a fluorescent signal associated with a biological activity.It is within ordinary skill in the art to modify such an instrument todetect a chromic signal.

In some embodiments, the system may further comprise a processor. Insome embodiments, the instrument may comprise a microprocessor capableof controlling the instrument and collecting and/or transmitting dataassociated with detecting the indicator reagent or biological derivativethereof. In some embodiments of the system, the processor may comprisean external processor. The external computer may comprise a personalcomputer (PC), desktop computer, laptop computer, handheld computer,workstation, or the like. For example, software programs can be loadedon external computer to control the instrument and/or to facilitate thecollection, transfer and/or analysis of data from the instrument.

In some embodiments, the system may further comprise means to regulatethe temperature of a liquid. The means for temperature control caninclude any means known in the art such as, for example, thermocouplesand heat-exchangers. Advantageously, these embodiments provide a systemthat can facilitate the biological activity by controlling thetemperature and can detect the product of the biological activity.

Biological Sterilization Indicators:

FIGS. 4-10 illustrate the biological sterilization indicator 100according to one embodiment of the present disclosure. Other suitableembodiments of biological sterilization indicators are described inInternational Publication Nos. WO 2011/011189, WO 2012/061229, WO2012/061228, and WO 2012/061227; each of which is incorporated herein byreference in its entirety.

The biological sterilization indicator 100 can include a housing 102,which can include a first portion 104 and a second portion 106 (e.g., acap) adapted to be coupled together to provide a self-containedbiological sterilization indicator. In some embodiments, the firstportion 104 and second portion 106 can be formed of the same materials,and in some embodiments, the first portion 104 and the second portion106 can be formed of different materials. The housing 102 can define areservoir 103 of the biological sterilization indicator 100 in whichother components can be positioned and into which a sterilant can bedirected during a sterilization process.

The housing 102 can be defined by at least one liquid impermeable wall,such as a wall 108 of the first portion 104 and/or a wall 110 of thesecond portion 106. It should be understood that a one-part unitaryhousing 102 may also be employed or that the first and second portions104 and 106 can take on other shapes, dimensions, or relative structureswithout departing from the spirit and scope of the present disclosure.Suitable materials for the housing 102 (e.g., the walls 108 and 110) caninclude, but are not limited to, a glass, a metal (e.g., foil), apolymer (e.g., polycarbonate (PC), polypropylene (PP), polyphenylene(PPE), polyethylene, polystyrene (PS), polyester (e.g., polyethyleneterephthalate (PET)), polymethyl methacrylate (PMMA or acrylic),acrylonitrile butadiene styrene (ABS), cyclo olefin polymer (COP), cycloolefin copolymer (COC), polysulfone (PSU), polyethersulfone (PES),polyetherimide (PEI), polybutyleneterephthalate (PBT)), a ceramic, aporcelain, or combinations thereof.

In some embodiments, the biological sterilization indicator 100 canfurther include a frangible container 120 that contains a liquid 122,and which is dimensioned to be received within the biologicalsterilization indicator 100, for example, within at least a portion ofthe housing 102 (e.g., at least within the first portion 104 of thehousing 102). The frangible container 120 can be formed of a variety ofmaterials, including, but not limited to, one or more of metal (e.g.,foil), a polymer (e.g., any of the polymers listed above with respect tothe housing 102), glass (e.g., a glass ampoule), and combinationsthereof. In some embodiments, only a portion of the container 120 isfrangible, for example, the container 120 can include a frangibleportion or cover (e.g., a frangible barrier, film, membrane, or thelike). The frangible container 120 can have a first state in which it isintact and the liquid 122 is contained therein, and a second state inwhich at least a portion of the container 120 is fractured. In thesecond state of the container 120, the liquid 122 can be in fluidcommunication with the reservoir 103 of the biological sterilizationindicator 100, e.g., when the container 120 is positioned in thebiological sterilization indicator 100.

As shown in the illustrated embodiment, the container 120 can be held inplace within the biological sterilization indicator 100 and/or fracturedby an insert 130, which is described in greater detail below. Thecontainer 120 can be fractured, for example, by urging the container 120against the insert 130 (e.g., an insert that functions as a breaker) orby urging the insert 130 against the container 120.

The first portion 104 of the housing 102 can be adapted to house amajority of the components of the biological sterilization indicator100, and can be referred to as a “tube,” “tubular body,” “base,” or thelike. The housing 102 can include a reservoir 103 that can be defined byone or both of the first portion 104 and the second portion 106 of thehousing 102. The biological sterilization indicator 100 can furtherinclude spores or another source(s) of biological activity 115 (or alocus of spores) positioned in fluid communication with the reservoir103. As shown in FIGS. 4-6, the second portion 106 of the housing 102can include one or more apertures 107 to provide fluid communicationbetween the interior of the housing 102 (e.g., the reservoir 103) andambience. For example, the one or more apertures 107 can provide fluidcommunication between the spores 115 and ambience during a sterilizationprocess, and can serve as an inlet into the biological sterilizationindicator 100 and as an inlet of a sterilant path 164 (described ingreater detail below). In some embodiments, the second portion 106 ofthe housing 102 can be coupled to a first (e.g., open) end 101 of thefirst portion 104 of the housing 102, and the spores 115 can bepositioned at a second (e.g., closed) end 105, opposite the first end101, of the first portion 104 of the housing 102.

In some embodiments, a barrier or filter (e.g., a sterile barrier; notshown) can be positioned in the sterilant path 164 (e.g., at the inletformed by the aperture 107) to inhibit contaminating or foreignorganisms, objects or materials from entering the biologicalsterilization indicator 100. Such a barrier can include agas-transmissive, microorganism-impermeable material, and can be coupledto the housing 102 by a variety of coupling means, including, but notlimited to, an adhesive, a heat seal, sonic welding, or the like.Alternatively, the barrier can be coupled to the sterilant path 164 viaa support structure (such as the second portion 106) that is coupled tothe first portion 104 of the housing 102 (e.g., in a snap-fitengagement, a screw-fit engagement, a press-fit engagement, or acombination thereof). During exposure to a sterilant, the sterilant canpass through the barrier into the sterilant path 164 and into contactwith the spores 115.

In some embodiments, as shown in the illustrated embodiment, the housing102 can include a lower portion 114 and an upper portion 116, which canbe at least partially separated by an inner wall (or partial wall) 118,ledge, partition, flange, or the like, in which can be formed an opening117 that provides fluid communication between the lower portion 114 andthe upper portion 116. In some embodiments, the lower portion 114 of thefirst portion 104 of the housing 102 (sometimes referred to as simply“the lower portion 114” or the “the lower portion 114 of the housing102”) can be adapted to house the spores 115 or a locus of spores. Insome embodiments, the lower portion 114 can be referred to as the“detection portion” or “detection region” of the housing 102, because atleast a portion of the lower portion 114 can be interrogated for signsof spore growth. In addition, in some embodiments, the upper portion 116of the first portion 104 of the housing 102 (sometimes referred to as“the upper portion 116” or the “the upper portion 116 of the housing102” for simplicity) can be adapted to house at least a portion of thefrangible container 120, particularly before activation.

In some embodiments, the portion of the reservoir 103 that is defined atleast partially by the upper portion 116 of the housing 102 can bereferred to as a first chamber (or reservoir, zone, region, or volume)109 and the portion of the reservoir 103 that is defined at leastpartially by the lower portion 114 of the housing 102 can be referred toas a second chamber (or reservoir, zone, region, or volume) 111. In someembodiments, the second chamber 111 can be referred to as a “sporegrowth chamber” or a “detection chamber,” and can include a volume to beinterrogated for spore viability to determine the efficacy of asterilization process.

The first chamber 109 and the second chamber 111 can be positioned influid communication with each other to allow a sterilant and the liquid122 to move from (i.e., through) the first chamber 109 to the secondchamber 111. In some embodiments, the degree of fluid connection betweenthe first chamber 109 and the second chamber 111 (e.g., the size of anopening, such as the opening 117, connecting the first chamber 109 andthe second chamber 111) can increase after, simultaneously with, and/orin response to the activation step (i.e., the liquid 122 being releasedfrom the container 120). In some embodiments, the control of fluidcommunication (or extent of fluid connection) between the first chamber109 (e.g., in the upper portion 116) and the second chamber 111 (e.g.,in the lower portion 114) can be provided by at least a portion of theinsert 130.

The container 120 can be positioned and held in the first chamber 109during sterilization and when the container 120 is in a first,unfractured, state. The spores 115 can be housed in the second chamber111 and in fluid communication with ambience when the container 120 isin the first state. The first chamber 109 and the second chamber 111 canbe configured such that the container 120 is not present in the secondchamber 111, and particularly, not when the container 120 is in itsfirst, unfractured, state. A sterilant can move into the second chamber111 (e.g., via the first chamber 109) during sterilization, and theliquid 122 can move into the second chamber 111 (e.g., from the firstchamber 109) during activation, when the container 120 is fractured andthe liquid 122 is released into the interior of the housing 102.

As a result, when the container 120 is in the first state, the firstchamber 109 and the second chamber 111 can be in fluid communicationwith one another, and with ambience (e.g., during sterilization). Forexample, the first chamber 109 and the second chamber 111 can be influid communication with ambience via the one or more apertures 107. Insome embodiments, the first chamber 109 and the second chamber 111 canbe in fluid communication with ambience in such a way that the firstchamber 109 is positioned upstream of the second chamber 111 when asterilant is entering the biological sterilization indicator 100. Thatis, the first chamber 109 can be positioned between the sterilant inlet(e.g., the one or more apertures 107) and the second chamber 111, andthe sterilant inlet can be positioned on an opposite side of the firstchamber 109 than the second chamber 111.

As shown in FIGS. 7 and 9, in some embodiments, the first chamber 109can be defined by one or both of the first portion 104 and the secondportion 106, particularly when the container 120 is in the first state.In addition, in some embodiments, the first chamber 109 can include afirst end 112 positioned adjacent the open end 101 of the first portion104 of the housing 102, adjacent the second portion 106 of the housing102, and/or at least partially defined by the second portion 106. Thefirst chamber 109 can further include a second end 13 positionedadjacent and in fluid communication with the second chamber 111 andpositioned toward the closed end 105 of the housing 102. The first end112 of the first chamber 109 can be at defined by the first portion 104and/or the second portion 106 of the housing 102.

As further shown in FIGS. 7 and 9, in some embodiments, the secondchamber 111 can include a first end 124 positioned adjacent and in fluidcommunication with the first chamber 109 and positioned toward the openend 101 of the housing 102, and a second end 125 at least partiallydefined by, including, or positioned adjacent the closed end 105 of thehousing 102.

Said another way, as shown in FIGS. 7 and 9, the biologicalsterilization indicator 100 can include a longitudinal direction D_(L),and in some embodiments, the first chamber 109 can be positionedlongitudinally above the second chamber 111.

In some embodiments, the second chamber 111 can be at least partiallydefined by, can include, or can be positioned adjacent the closed end105 of the biological sterilization indicator 100. In addition, in someembodiments, the second chamber 111 can be smaller (e.g., in volumeand/or cross-sectional area) than at least one of the first chamber 109and the volume of the liquid 122 in the container 120 that will bereleased when the biological sterilization indicator 100 is activated.As a result, in such embodiments, the second chamber 111 can exhibit anair-lock effect where gas (e.g. air) that is present in the secondchamber 111 can inhibit fluid movement into the second chamber 111. Insome embodiments, as described in greater detail below, a fluid paththat allows the second chamber 111 to vent to another portion of thebiological sterilization indicator 100 can facilitate fluid movementinto the second chamber 111.

In some embodiments, the wall 118 (sometimes referred to as a“separating wall”) can be angled or slanted, for example, oriented at anon-zero and non-right angle with respect to the longitudinal directionD_(L) of the housing 102 (e.g., where the longitudinal direction D_(L)extends along the length of the housing 102). Such angling or slantingof the wall 118 can facilitate the movement of the liquid 122 from theupper portion 116 to the lower portion 114 after sterilization and afterthe container 120 has been broken to release the liquid 122.

As shown in FIGS. 4-6, in some embodiments, the wall 118 can be at leastpartially formed by a change in the inner dimension of the housing 102.For example, as shown, the wall 118 can be formed by a decrease in across-sectional area from a first longitudinal position in the firstchamber 109 to a second longitudinal position in the second chamber 111.In addition, by way of example only, the internal cross-sectional shapeof the housing 102 can change at the transition from the first chamber109 to the second chamber 111 from being substantially round (e.g., withone flat side that makes up less than 50% of the perimeter) in the firstchamber 109 to substantially parallelepipedal (e.g., substantiallysquare) in the second chamber 111.

Furthermore, in some embodiments, the wall 118 can also be at leastpartially formed by a change in the outer dimension of the housing 102.As shown in FIGS. 4-6, in some embodiments, the housing 102 includes astep (or ledge, overhang, transition, or the like) 123 that is angledconsistently with the wall 118 (if the wall 118 is angled), and whichincludes a change in the outer shape and dimension of the housing 102.However, it should be understood that in some embodiments, even if theinner dimension of the housing 102 changes to create a second chamber111 that has a different cross-sectional shape or dimension than thefirst chamber 109, the outer shape and dimension of the housing 102 neednot change, or change consistently with the change in the inner shapeand/or dimension. For example, in some embodiments, the step 123 can beoriented substantially perpendicularly with respect to the longitudinaldirection D_(L).

In some embodiments, the reservoir 103 has a volume of at least about0.5 milliliters (mL), in some embodiments, at least about 1 mL, and insome embodiments, at least about 1.5 mL. In some embodiments, thereservoir 103 has a volume of no greater than about 5 mL, in someembodiments, no greater than about 3 mL, and in some embodiments, nogreater than about 2 mL.

In some embodiments, the frangible container 120 has a volume of atleast about 0.25 mL, in some embodiments, at least about 0.5 mL, and insome embodiments, at least about 1 mL. In some embodiments, thefrangible container 120 has a volume of no greater than about 5 mL, insome embodiments, no greater than about 3 mL, and in some embodiments,no greater than about 2 mL.

In some embodiments, the volume of the liquid 122 contained in thefrangible container 120 is at least about 50 microliters, in someembodiments, at least about 75 microliters, and in some embodiments, atleast about 100 microliters. In some embodiments, the volume of theliquid 122 contained in the frangible container 120 is no greater thanabout 5 mL, in some embodiments, no greater than about 3 mL, and in someembodiments, no greater than about 2 mL.

In some embodiments, the first chamber 109 (i.e., formed by the upperportion 116 of the first portion 104 of the housing 102) has a volume ofat least about 500 microliters (or cubic millimeters), in someembodiments, at least about 1000 microliters, in some embodiments, atleast about 2000 microliters, and in some embodiments, at least about2500 microliters. In some embodiments, the first chamber 109 has avolume of no greater than about 5000 microliters, in some embodiments,no greater than about 4000 microliters, and in some embodiments, nogreater than about 3000 microliters. In some embodiments, the firstchamber 109 has a volume of about 2790 microliters, or 2800 microliters.

In some embodiments, the second chamber 111 (i.e., formed by the lowerportion 114 of the first portion 104 of the housing 102) has a volume ofat least about 5 microliters, in some embodiments, at least about 20microliters, and in some embodiments, at least about 35 microliters. Insome embodiments, the second chamber 111 has a volume of no greater thanabout 250 microliters, in some embodiments, no greater than about 200microliters, in some embodiments, no greater than about 175 microliters,and in some embodiments, no greater than about 100 microliters. In someembodiments, the second chamber 111 has a volume of about 208microliters, or 210 microliters.

In some embodiments, the volume of the second chamber 111 is at leastabout 5% of the volume of the first chamber 109, and in someembodiments, at least about 7%. In some embodiments, the volume of thesecond chamber 111 is no greater than about 20% of the volume of thefirst chamber 109, in some embodiments, no greater than about 15%, insome embodiments, no greater than about 12%, and in some embodiments, nogreater than about 10%. In some embodiments, the volume of the secondchamber 111 is about 7.5% of the volume of the first chamber 109.

In some embodiments, the volume of the second chamber 111 is no greaterthan about 60% of the volume of the liquid 122 housed in the container120, in some embodiments, no greater than about 50%, and in someembodiments, no greater than about 25%. In some embodiments, designingthe second chamber 111 to have a volume that is substantially less thanthat of the liquid 122 housed in the container 120 can ensure that theadditional liquid volume can compensate for unintended evaporation.

In some embodiments, the first chamber 109 (i.e., formed by the upperportion 116 of the first portion 104 of the housing 102) has across-sectional area (or average cross-sectional area) at the transitionbetween the first chamber 109 and the second chamber 111, or at theposition adjacent the second chamber 111, of at least about 25 mm²; insome embodiments, at least about 30 mm²; and in some embodiments, atleast about 40 mm². In some embodiments, the first chamber 109 has across-sectional area at the transition between the first chamber 109 andthe second chamber 111, or at the position adjacent the second chamber111, of no greater than about 100 mm², in some embodiments, no greaterthan about 75 mm², and in some embodiments, no greater than about 50mm².

In some embodiments, the second chamber 111 (i.e., formed by the lowerportion 114 of the first portion 104 of the housing 102) has across-sectional area at the transition between the first chamber 109 andthe second chamber 111, or at the position adjacent the first chamber109, of at least about 5 mm², in some embodiments, at least about 10mm², and in some embodiments, at least about 15 mm². In someembodiments, the second chamber 111 has a cross-sectional area (oraverage cross-sectional area) of no greater than about 30 mm², in someembodiments, no greater than about 25 mm², and in some embodiments, nogreater than about mm².

In some embodiments, the cross-sectional area of the second chamber 111at the transition between the first chamber 109 and the second chamber111 can be no greater than about 60% of the cross-sectional area of thefirst chamber 109 at the transition, in some embodiments, no greaterthan about 50%, in some embodiments, no greater than about 40%, and insome embodiments, no greater than about 30%.

In some embodiments, the biological sterilization indicator 100 canfurther include a substrate 119. In some embodiments, as shown in FIGS.4-7 and 9, the substrate 119 can be dimensioned to be positionedadjacent the wall 118, and particularly, to rest atop the wall 118. Thesubstrate 119 can be positioned between the upper portion 116 (i.e., thefirst chamber 109) and the lower portion 114 (i.e., the second chamber111) of the biological sterilization indicator 100 and, in someembodiments, can at least partially define the first chamber 109 and thesecond chamber 111. As such, in some embodiments, the substrate 119 canbe positioned between the container 120 and the spores 115. In someembodiments, the substrate 119 can be positioned in the first chamber109, or on a first chamber side of the wall 118, such that the substrate119 is not positioned in the second chamber 111.

In addition, the substrate 119 can be positioned to minimize diffusionof an assay signal (e.g., fluorescence) out of the second chamber 111.In some embodiments, depending on the material makeup of the substrate119, the substrate 119 can also absorb dyes, indicator reagents, orother materials from solution that may inhibit accurate reading of asignal from the biological sterilization indicator 100 (i.e.,“inhibitors”). In some embodiments, as shown in FIGS. 4-7, 9 and 10, thesubstrate 119 can include one or more apertures 121, which can beconfigured to control (i.e., facilitate and/or limit, depending onnumber, size, shape, and/or location) fluid movement between the firstchamber 109 and the second chamber 111 of the biological sterilizationindicator 100, and particularly, which can facilitate movement of theliquid 122 to the spores 115 when the container 120 is fractured. By wayof example only, particular benefits or advantages were observed whenthe aperture 121 was positioned front of (or “forward of”) the center ofthe substrate 119, as shown. In the embodiment illustrated in FIGS.4-10, the “front” of the biological sterilization indicator 100 orcomponents therein can generally be described as being toward a flatface 126. In general, the “front” of the biological sterilizationindicator 100 can refer to the portion of the biological sterilizationindicator 100 that will be interrogated by a reading apparatus.

In addition, by way of example only, the aperture 121 is illustrated asbeing circular or round; however, other cross-sectional aperture shapesare possible and within the scope of the present disclosure.Furthermore, by way of example only, and as shown in FIG. 6, thesubstrate 119 is shaped to substantially fill the first chambercross-sectional area at the transition between the first chamber 109 andthe second chamber 111. However, other shapes of the substrate 119 arepossible and can be adapted to accommodate the housing 102, the firstchamber 109, the second chamber 111, the wall 118, or another componentof the biological sterilization indicator 100.

As mentioned above, the second chamber 111 can include a volume to beinterrogated. Such a volume can be assayed for spore viability todetermine the lethality or effectiveness of a sterilization procedure.In some embodiments, the volume to be interrogated can be all or aportion of the second chamber 111. In some embodiments, the substrate119 can be positioned outside of the volume to be interrogated, whichcan minimize the number of structures in the volume that may interferewith the assaying processes. For example, in some embodiments, thesubstrate 119 can be positioned such that the substrate 119 is not indirect contact with at least one of the spores 115, the spore carrier135, and the spore reservoir 136. In some embodiments, the substrate 119can be positioned such that the substrate 119 is not located between adetection system (e.g., an optical detection system, such as afluorescence excitation source and an emission detector) and at leastone of the spores 115, the spore carrier 135, and the spore reservoir136. The substrate 119 can have the above positions when the container120 is in the first state and/or the second state, but particularly,when the container 120 is in the second state.

In some embodiments, substrate position in the biological sterilizationindicator 100 can affect the correlation of a rapid detection system forspore viability (e.g., fluorescence detection) with a slower (e.g.,overnight or 24-hr) detection system (e.g., a pH indicator that canexhibit a color change (e.g., in 24 hr) in response to spore growth).For example, in some embodiments, the substrate 119 can improve thecorrelation of fluorescence readings at various timepoints with growthresults after 24 hrs. Particularly, when the substrate 119 is positionedin a “first” position—as described herein and as shown in FIGS. 1, 2,and 4-7—the fluorescence can accurately correlate to growth. Suchcorrelation can be an improvement over other substrate positions andbiological sterilization indicators with no substrate.

In addition, the substrate 119 can be positioned in the biologicalsterilization indicator 100 such that the substrate 119 is not in directcontact with the container 120 when the container 120 is in the firststate. For example, in some embodiments, the substrate 119 can bepositioned in the first chamber 109 (e.g., adjacent a bottom end (e.g.,the second end 113) of the first chamber 109), but even in suchembodiments, the substrate 119 can be positioned such that the substrate119 does not contact the container 120. For example, as shown in FIGS.4-5 and 7-9, in some embodiments, the insert 130 can be positionedbetween the container 120 and the substrate 119 when the container 120is in the first state, such that the insert 130 holds the container 120in the first state. The insert 130, or a portion thereof, can bepositioned adjacent the substrate 119. For example, as shown in theillustrated embodiment, the substrate 119 can be positioned between(e.g., sandwiched between) the insert 130 and the wall 118. As such, insome embodiments, the substrate 119 can be positioned between the insert130 and the second chamber 111.

As mentioned above, in some embodiments, the substrate 119 can bepositioned and configured to control or affect fluid flow in thebiological sterilization indicator 100, and particularly, to controlfluid flow between the first chamber 109 and the second chamber 111. Forexample, in some embodiments, the substrate 119 can be configured (e.g.,sized, shaped, oriented, and/or constructed of certain materials) tocontrol the rate at which a sterilant is delivered to the second chamber111 (and to the spores 115). For example, the sterilant delivery ratecan be less than it otherwise would be if the substrate 119 were notpresent between the first chamber 109 and the second chamber 111.

Furthermore, in some embodiments, the substrate 119 can be configured(e.g., sized, shaped, positioned, oriented, and/or constructed ofcertain materials) to control the rate at which detectable productsdiffuse out of the volume to be interrogated. In some embodiments, thedetectable product can include a signal (e.g., a fluorescent signal)that indicates spore viability, and in some embodiments, the detectableproduct can be the spore(s) 115 itself. Controlling the diffusion ofdetectable products out of the volume to be interrogated can beparticularly useful in embodiments in which the volume of the liquid 122is greater than the volume of the second chamber 111 (or of the volumeto be interrogated), because the liquid 112 in such embodiments canextend in the biological sterilization indicator 100 to a higher levelthan the second chamber 111 (or the volume to be interrogated) when thecontainer 120 is in its second, fractured, state. In such embodiments,detectable products can be free to move throughout the full volume ofthe liquid 122 (i.e., to a volume outside of the volume to beinterrogated), unless there is some barrier or means for controllingsuch diffusion, such as the substrate 119. For example, in someembodiments, the substrate 119 can be positioned at a level just abovethe volume to be interrogated (i.e., below the level of the liquid 122),to inhibit movement of the detectable products to the portion of theliquid 122 that is positioned above the substrate 119.

In some embodiments, the substrate 119 can control sterilant deliveryrate (e.g., into the second chamber 111) and/or the diffusion rate ofdetectable products (e.g., out of the second chamber 111) by providing aphysical barrier or blockage to the sterilant and/or the detectableproducts. Such a physical barrier can also function to collect brokenportions of the container 120 when the container 120 is in the second,fractured, state to inhibit movement of the broken portions into thevolume to be interrogated where the broken portions could block,refract, reflect, or otherwise interfere with detection processes (e.g.,optical detection processes).

In addition, in some embodiments, the liquid 122, either before or aftercoming into fluid communication with the spores 115, can include one ormore inhibitors, or other components, that may interfere with anaccurate assay or detection process. In some embodiments, examples ofinhibitors can include at least one of dyes, indicator reagents, othermaterials or substances that may inhibit a reaction (e.g., an enzymaticreaction) necessary for detection of spore viability (e.g., salts,etc.), other materials or substances that may interfere with thedetection process, or combinations thereof. In such embodiments, thesubstrate 119 can be configured to absorb and/or selectively concentrateone or more inhibitors from the liquid 122, or at least from the volumeof the liquid 122 to be interrogated.

For example, in some embodiments, more than one indicator reagent can bepresent in the liquid 122, either before contacting the spores 115 or asa result of contacting the spores 115. In such embodiments, while afirst indicator reagent (e.g., used for fluorescence detection) may benecessary for spore viability detection, a second indicator reagent(e.g., a pH indicator) may actually interfere with the detection of thefirst indicator reagent. By way of example only, in embodiments in whichthe second indicator reagent is a pH indicator (e.g., one or more ofbromocresol purple, methyl red, or a combination thereof), the pHindicator may conflict or interfere with the fluorescence reading of thefirst indicator reagent, for example, in embodiments in which the pHindicator emits electromagnetic radiation at a wavelength that issimilar to the spectral band of the fluorescence of the first indicatorreagent (e.g., when the pH indicator exhibits a color shift). In suchembodiments, the substrate 119 can be configured (e.g., formed of anappropriate material) to absorb and/or selectively concentrate thesecond indicator reagent when positioned in contact with the liquid 122to reduce the concentration of the second indicator reagent in theliquid 122, or at least in the volume of the liquid 122 to beinterrogated.

In addition, in some embodiments (e.g., in embodiments in which the wall118 is slanted and the substrate 119 is positioned adjacent the wall118), the substrate 119 can be angled or slanted, for example, orientedat a non-zero and non-right angle with respect to the longitudinaldirection D_(L) of the housing 102. Such angling or slanting of thesubstrate 119 can facilitate the movement of the liquid 122 from thefirst chamber 109 to the second chamber 111 after sterilization andafter the container 120 has been broken to release the liquid 122.

In some embodiments, the substrate 119 can be formed of a variety ofmaterials to accomplish one or more of the above functions. Examples ofsubstrate materials can include, but are not limited to, cotton, glasswool, cloth, nonwoven polypropylene, nonwoven rayon, nonwovenpolypropylene/rayon blend, nonwoven nylon, nonwoven glass fiber or othernonwoven fibers, filter papers, microporous hydrophobic and hydrophilicfilms, glass fibers, open celled polymeric foams, and semi-permeableplastic films (e.g., particle filled films, thermally induced phaseseparation (TIPS) membranes, etc.), and combinations thereof. Forexample, in embodiments in which the substrate 119 can be used toselectively concentrate one more indicator reagents (e.g., bromocresolpurple (BCP)), the substrate 119 can be formed of a charged nylon (suchthe reprobing, charged transfer membranes available from GE Osmonics(under the trade designation “MAGNAPROBE” (e.g., 0.45 micron, CatalogNo. NP0HY00010, Material No. 1226566)).

An example of a method and system that can employ the substrate 119 isalso described in International Patent Publication No. WO 2012/061212,which is incorporated herein by reference in its entirety.

In some embodiments, at least a portion of one or more of the insert130, the wall 118, and/or the substrate 119, or an opening therein, canprovide fluid communication between the first chamber 109 (e.g., in theupper portion 116) and the second chamber 111 (e.g., in the lowerportion 114), and/or can control the fluid communication between thefirst chamber 109 and the second chamber 111 (e.g., by controlling theextent of fluid connection between the first chamber 109 and the secondchamber 111).

The biological sterilization indicator 100 can include a first fluidpath 160 that can be positioned to fluidly couple the first chamber 109and the second chamber 111, and which can allow sterilant (e.g., duringsterilization, when the container 120 is in a first, unfractured, state)and/or the liquid 122 (e.g., after sterilization and during activation,when the container 120 is in a second, fractured, state) to reach thespores 115. In the illustrated embodiment the first fluid path 160 cangenerally be defined by one or more of the following: (1) the insert130, e.g., via an aperture 177 described below, an opening formed in theinsert 130, and/or any open spaces around the insert 130, such asbetween the insert 130 (e.g., a front portion thereof) and the housing102; (2) the wall 118, e.g., the aperture 117 defined by the wall 118;(3) the substrate 119, e.g., the aperture 121 formed therein, or anyopen spaces around the substrate 119, such as between the substrate 119(e.g., a front portion thereof) and the housing 102; (4) the housing102, e.g., any openings or spaces formed therein; and combinationsthereof. As a result, the first fluid path 160 is generally representedin the illustrated embodiment by an arrow in FIGS. 7 and 10.

The biological sterilization indicator 100 can further include a secondfluid path 162 positioned to fluidly couple the second chamber 111 withanother chamber or portion of the biological sterilization indicator100, such as the first chamber 109. The second fluid path 162 can befurther positioned to allow gas that was previously present in thesecond chamber 111 to be displaced and to exit the second chamber 111,for example, when the sterilant and/or the liquid 122 is moved into thesecond chamber 111. As such, the second fluid path 162, which isdescribed in greater detail below, can serve as an internal vent in thebiological sterilization indicator 100.

In some embodiments, the substrate 119 can provide a physical barrier orblockage between the first chamber 109 and the second chamber 111 whichcan allow for at least one of the following: controlling the sterilantdelivery rate/kill rate at which sterilant is delivered into the secondchamber 111; controlling the diffusion of spores 115 and/or detectableproducts out of the second chamber 111; controlling the delivery rate ofthe liquid 122 to the second chamber 111 (and to the spores 115) whenthe container 120 is in the second, fractured, state; or a combinationthereof.

Because, in some embodiments, the substrate 119 can provide a physicalbarrier to delivering the liquid 122 to the second chamber 111 duringactivation (i.e., when the container 120 is in the second state),aperture 121 in the substrate 119 and/or the angle of the substrate 119can be controlled to effect a desired liquid delivery rate. In addition,or alternatively, the second fluid path 162 can provide a vent for anygas or air that is trapped in the second chamber 111 to facilitatemoving the liquid 122 through or past the substrate 119 and into thesecond chamber 111 when desired.

In addition, or alternatively, the housing 102 can be configured (e.g.,formed of an appropriate material and/or configured with microstructuredgrooves or other physical surface modifications) to facilitate movingthe liquid 122 to the second chamber 111 when desired.

In some embodiments, the liquid 122 can include a nutrient medium forthe spores, such as a germination medium that will promote germinationof surviving spores. In some embodiments, the liquid 122 can includewater (or another solvent) that can be combined with nutrients to form anutrient medium. Suitable nutrients can include nutrients necessary topromote germination and/or growth of surviving spores and may beprovided in a dry form (e.g., powdered form, tablet form, caplet form,capsule form, a film or coating, entrapped in a bead or other carrier,another suitable shape or configuration, or a combination thereof) inthe reservoir 103, for example, in a region of the biologicalsterilization indicator 100 near the spores 115.

The nutrient medium can generally be selected to induce germination andinitial outgrowth of the spores, if viable. The nutrient medium caninclude one or more sugars, including, but not limited to, glucose,fructose, cellobiose, or the like, or a combination thereof. Thenutrient medium can also include a salt, including, but not limited to,potassium chloride, calcium chloride, or the like, or a combinationthereof. In some embodiments, the nutrient can further include at leastone amino acid, including, but not limited to, at least one ofmethionine, phenylalanine, and tryptophan.

In some embodiments, the nutrient medium can include indicatormolecules, for example, indicator molecules having optical propertiesthat change in response to germination or growth of the spores. Suitableindicator molecules can include, but are not limited to, pH indicatormolecules, enzyme substrates, DNA binding dyes, RNA binding dyes, othersuitable indicator molecules, or a combination thereof.

As shown in FIGS. 4-10, the biological sterilization indicator 100 canfurther include an insert 130. In some embodiments, the insert 130 canbe adapted to hold or carry the container 120, such that the container120 is held intact in a location separate from the spores 115 duringsterilization. That is, in some embodiments, the insert 130 can include(or function as) a carrier 132 (see FIG. 4) for the container 120,particularly, before the container 120 is broken during the activationstep (i.e., the step in which the liquid 122 is released from thecontainer 120 and introduced to the spores 115, which can occur after asterilization process). In some embodiments, the insert 130 can befurther adapted to allow the container 120 to move at least somewhat inthe housing 102, e.g., longitudinally with respect to the housing 102.The insert 130 of the illustrated embodiment is described in greaterdetail below. Examples of other suitable inserts and carriers aredescribed in International Publication No. WO 2011/011189.

In some embodiments, the biological sterilization indicator 100 canfurther include a spore carrier 135, as shown in FIGS. 4-7 and 9.However, in some embodiments, the insert 130 can be modified to includea portion adapted to house the spores 115. For example, in someembodiments, the insert 130 and the spore carrier 135 can be integrallyformed as one insert comprising a first portion adapted to hold andeventually fracture the container 120, when desired, and a secondportion adapted to house the spores 115 in a region of the biologicalsterilization indicator 100 that is separate from the container 120during sterilization (i.e., prior to fracture).

As shown in FIGS. 4-7 and 9, the spore carrier 135 can include a sporereservoir 136 (which can also be referred to as a depression, divot,well, recess, or the like), in which the spores 115 can be positioned,either directly or on a substrate. In embodiments employing a nutrientmedium that is positioned to be mixed with the liquid 122 when it isreleased from the container 120, the nutrient medium can be positionednear or in the spore reservoir 136, and the nutrient medium can be mixedwith (e.g., dissolved in) the water when the water is released from thecontainer 120. By way of example only, in embodiments in which thenutrient medium is provided in a dry form, the dry form can be presentwithin the reservoir 103, the spore reservoir 136, on a substrate forthe spores, or a combination thereof. In some embodiments, a combinationof liquid and dry nutrient media can be employed.

In some embodiments, the spore reservoir 136 has a volume of at leastabout 1 microliter, in some embodiments, at least about 5 microliters,and in some embodiments, at least about 10 microliters. In someembodiments, the spore reservoir 136 has a volume of no greater thanabout 250 microliters, in some embodiments, no greater than about 175microliters, and in some embodiments, no greater than about 100microliters.

As shown in FIGS. 7 and 9, in some embodiments, the biologicalsterilization indicator 100 can further include a rib or protrusion 165that can be coupled to or integrally formed with a wall 108 of thehousing 102, which can be positioned to maintain the spore carrier 135in a desired location in the housing 102 and/or at a desired angle ororientation, for example, with respect to detection systems (e.g.,optical detection systems) of the reading apparatus 12.

As shown in FIGS. 4-7 and 9, the second portion 106 of the housing 102can be adapted to be coupled to the first portion 104. For example, asshown, the second portion 106 can be adapted to be coupled to the upperportion 116 (e.g., the first end 101) of the first portion 104 of thehousing 102. In some embodiments, as shown in FIGS. 4-7, the secondportion 106 can be in the form of a cap that can be dimensioned toreceive at least a portion of the first portion 104 of the housing 102.

As shown in FIGS. 4-5 and 7-8, during sterilization and beforeactivation, the second portion 106 can be in a first “unactivated”position 148 with respect to the first portion 104, and the container120 can be in a first, intact, state. As shown in FIG. 9, the secondportion 106 of the housing 102 can be moved to a second “activated”position 150 (e.g., where the second portion 106 is fully depressed)with respect to the first portion 104, and the container 120 can be in asecond, fractured, state. For example, after sterilization, thebiological sterilization indicator 100 can be activated by moving thesecond portion 106 from the first position 148 to the second position150 (i.e., a sufficient amount) to cause fracturing of the container 120and to release the liquid 122 from the container 120, to allow theliquid 122 to be in fluid communication with the spores 115. Thebiological sterilization indicator 100 can be activated prior topositioning the biological sterilization indicator 100 in a well of areading apparatus, after positioning the biological sterilizationindicator 100 in the well, or as the biological sterilization indicator100 is positioned in the well (i.e., the biological sterilizationindicator 100 can be slid into place in the reading apparatus, and thesecond portion 106 can continue to be pressed until it is in its secondposition 150, e.g., in which the bottom of the well provides sufficientresistance to move the second portion 106 to its second position 150).The second position 150 can be located closer to the closed end 105 ofthe first portion 104 of the biological sterilization indicator 100 thanthe first position 148.

As shown in the illustrated embodiment, in some embodiments, the firstportion 104 of the housing 102 can include a step, overhang, orflat-to-round transition 152. The step 152 is shown as being exposedwhen the second portion 106 is in its first position 148 and as beingobscured or covered when the second portion 106 is in its secondposition 150. As such, the step 152 can be detected to determine whetherthe second portion 106 is in the first position 148 (i.e., thebiological sterilization indicator 100 is unactivated), or is in thesecond position 150 (i.e., the biological sterilization indicator 100 isactivated). Using such features of the biological sterilizationindicator 100 to determine a status of the biological sterilizationindicator 100, for example, to confirm whether the biologicalsterilization indicator 100 has been activated, is described in greaterdetail in co-pending U.S. Application No. 61/409,042. The longitudinalposition of the step 152 is shown by way of example only; however, itshould be understood that the step 152 can instead be located at adifferent longitudinal position (e.g., closer to the closed end 105 ofthe biological sterilization indicator 100), or, in some embodiments,the transition from a rounded portion to a flat face can be gradual,tapered, or ramped.

A variety of coupling means can be employed between the first portion104 and the second portion 106 of the housing 102 to allow the firstportion 104 and the second portion 106 to be removably coupled to oneanother, including, but not limited to, gravity (e.g., one component canbe set atop another component, or a mating portion thereof), screwthreads, press-fit engagement (also sometimes referred to as“friction-fit engagement” or “interference-fit engagement”), snap-fitengagement, magnets, adhesives, heat sealing, other suitable removablecoupling means, and combinations thereof. In some embodiments, thebiological sterilization indicator 100 need not be reopened and thefirst portion 104 and the second portion 106 need not be removablycoupled to one another, but rather can be permanently orsemi-permanently coupled to one another. Such permanent orsemi-permanent coupling means can include, but are not limited to,adhesives, stitches, staples, screws, nails, rivets, brads, crimps,welding (e.g., sonic (e.g., ultrasonic) welding), any thermal bondingtechnique (e.g., heat and/or pressure applied to one or both of thecomponents to be coupled), snap-fit engagement, press-fit engagement,heat sealing, other suitable permanent or semi-permanent coupling means,and combinations thereof. One of ordinary skill in the art willrecognize that some of the permanent or semi-permanent coupling meanscan also be adapted to be removable, and vice versa, and are categorizedin this way by way of example only.

As shown in FIGS. 7 and 9, the second portion 106 can be movable betweena first longitudinal position 148 with respect to the first portion 104and a second longitudinal position 150 with respect to the first portion104; however, it should be understood that the biological sterilizationindicator 100 could instead be configured differently, such that thefirst and second positions 148 and 150 are not necessarily longitudinalpositions with respect to one or both of the first portion 104 and thesecond portion 106 of the housing 102.

The second portion 106 can further include a seal 156 (e.g., aprojection, a protrusion, a flap, flange, o-ring, or the like, orcombinations thereof) that can be positioned to contact the first end101 of the first portion 104, and particularly, an open upper end 157 ofthe first portion 104 to close or seal (e.g., hermetically seal) thebiological sterilization indicator 100 after the second portion 106 hasbeen moved to the second position 150 and the liquid 122 has beenreleased from the container 120 (i.e., when the container 120 is in asecond, fractured, state). That is, the spores 115 can be sealed fromambience when the container 120 is in the second state. The seal 156 cantake a variety of forms and is shown in FIGS. 7 and 9 by way of exampleas forming an inner ring or cavity that together with the wall 110 ofthe second portion 106 is dimensioned to receive the upper end 157 ofthe first portion 104 of the housing 102 to seal the biologicalsterilization indicator 100.

In some embodiments, one or both of the seal 156 and the upper end 157can further include a structure (e.g., a protrusion) configured toengage the other of the upper end 157 and the seal 156, respectively, inorder to couple the second portion 106 of the housing 102 to the firstportion 104 of the housing 102.

In addition, in some embodiments, the second portion 106 of the housing102 can be coupled to the first portion 104 of the housing 102 to sealthe biological sterilization indicator 100 from ambience afteractivation. Such sealing can inhibit contamination, evaporation, orspilling of the liquid 122 after it has been released from the container120, and/or can inhibit contamination of the interior of the biologicalsterilization indicator 100.

The insert 130 will now be described in greater detail.

As shown in FIGS. 4-5 and 7, during sterilization and before activation,the second portion 106 can be in a first position 148 with respect tothe first portion 104. In the first position 148, the container 120 canbe held intact in a position separate from the lower portion 114, thesecond chamber 111, or the spores 115, and the liquid 122 can becontained within the container 120.

As shown in FIG. 9, after sterilization, the biological sterilizationindicator 100 can be activated to release the liquid 122 from thecontainer 120 to move the liquid 122 to the second chamber 111. That is,the second portion 106 of the housing 102 can be moved to a secondposition 150 with respect to the first portion 104. When the secondportion 106 is moved from the first position 148 to the second position150, the seal 156 of the second portion 106 of the housing 102 canengage the upper end 157 of the first portion 104 to seal the reservoir103 of the biological sterilization indicator 100 from ambience. In suchembodiments, the second portion 106 can reversibly engage the firstportion 104 in the second position 150, and in some embodiments, thesecond portion 106 can irreversibly engage the first portion 104.However, it should be understood that the structures and coupling meansfor the first portion 104 and the second portion 106 are shown inillustrated embodiment by way of example only, and any of theabove-described coupling means can instead be employed between the firstportion 104 and the second portion 106 of the housing 102.

The insert 130 can be adapted to hold or carry the container 120, suchthat the container 120 is held intact in a location separate from thespores 115 during sterilization. That is, as mentioned above, in someembodiments, the insert 130 can include (or function as) a carrier 132for the container 120, particularly, before the container 120 is brokenduring the activation step (i.e., the step in which the liquid 122 isreleased from the container 120 and introduced to the spores 115, whichtypically occurs after a sterilization process).

In addition, the insert 130 can be adapted to hold the container 120intact in a position in the housing 102 that maintains at least aminimal spacing (e.g., a minimal cross-sectional area of space) betweenthe container 120 and the housing 102 and/or between the container 120and any other components or structures in the housing 102 (e.g., atleast a portion of the insert 130, such as the carrier 132, etc.), forexample, to maintain a substantially constant sterilant path 164 in thebiological sterilization indicator 100. In some embodiments, the insert130 can be adapted to hold the container 120 in a substantiallyconsistent location in the housing 102.

In some embodiments, as shown in FIG. 6, at least a portion of thehousing 102 can include a tapered portion 146 in which the housing 102(e.g., the wall 108 and/or an inner surface thereof) generally tapers inthe longitudinal direction D_(L) of the housing 102. As a result, thecross-sectional area in the housing 102 can generally decrease along thelongitudinal direction D_(L).

In some cases, without providing the means to maintain at least aminimal spacing around the container 120 (e.g., between the container120 and surrounding structure), there can be a possibility that thecontainer 120 can become positioned in the housing 102 (e.g., in thetapered portion 146) in such a way that it obstructs or blocks thesterilant path 164. However, the biological sterilization indicator 100of the present disclosure is designed to inhibit this from occurring.For example, in the illustrated embodiment, the insert 130 (andparticularly, the carrier 132) can be configured to hold the container120 out of the tapered portion 146 of the housing 102, such that atleast a minimal cross-sectional area is maintained around the container120 in any orientation of the biological sterilization indicator 100prior to activation. For example, in the embodiment illustrated in FIGS.4-8, even if the biological sterilization indicator 100 is tipped upsidedown, the container 120 may fall away from contact with the insert 130,but in no orientation, is the container 120 moved any closer to thetapered portion 146, or the spores 115 until activation of thebiological sterilization indicator 100. In addition, until activation,at least a minimal spacing (and particularly, a cross-sectional area ofthat spacing) between the container 120 and the housing 102 and/or theinsert 130 can be maintained to provide a substantially constantsterilant path 164, for example, around the container 120, through thefirst fluid path 160 and into the second chamber 111.

In some embodiments, the relative sizing and positioning of thecomponents of the biological sterilization indicator 100 can beconfigured such that, before activation, the container 120 is heldintact in a substantially consistent location in the biologicalsterilization indicator 100. Such a configuration can provide asubstantially constant sterilant path 164 and can maintain the container120 in a position such that the container 120 is not able to movesubstantially, if at all, in the biological sterilization indicator 100before activation.

In some embodiments, at least a portion of the insert 130 can be adaptedto allow the container 120 to move in the housing 102, e.g.,longitudinally with respect to the housing 102, between a first(longitudinal) position in which the container 120 is intact and asecond (longitudinal) position in which at least a portion of thecontainer 120 is fractured. By way of example only, the insert 130 caninclude one or more projections or arms 158 (two projections 158 spacedabout the container 120 are shown by way of example only) adapted tohold and support the container 120 before activation and to allow thecontainer 120 to move in the housing 102 during activation, for example,when the second portion 106 is moved with respect to the first portion104 of the housing 102. The projections 158 can also be adapted (e.g.,shaped and/or positioned) to fracture the container 120 in a desiredmanner when the biological sterilization indicator is activated. As aresult, the insert 130 can sometimes function to hold the container 120intact before activation, and can function to break the container 120during activation. As a result, the insert 130, or a portion thereof,can sometimes be referred to as a “carrier” (e.g., the carrier 132)and/or a “breaker.”

By way of example only, the projections 158 are shown in FIGS. 4 and6-10 as being coupled to a base or support 127 adapted to abut theseparating wall 118. For example, the base 127 can be dimensioned to bereceived in the reservoir 103 and dimensioned to sit atop, abut, orotherwise cooperate with or be coupled to the separating wall 118. Suchcoupling with an internal structure of the biological sterilizationindicator 100 can provide the necessary resistance and force to breakthe container 120 when desired. In some embodiments, however, the insert130 does not include the base 127, and the projections 158 can becoupled to or form a portion of the housing 102. In some embodiments,the insert 130 is integrally formed with or provided by the housing 102.

As shown, the insert 130 can further include a sidewall 131 thatconnects the projections 158 and is shaped to accommodate an innersurface of the housing 102 and/or an outer surface of the container 120.Such a sidewall 131 can provide support and rigidity to the projections158 to aid in reliably breaking the container 120 in a consistentmanner. The sidewall 131 can also be shaped and dimensioned to guide thecontainer 120 in a desired manner as it is moved in the housing 102during activation, for example, to contact the projections 158 in adesired way to reliably fracture the container 120. The sidewall 131and/or the wall 108 of the housing 102 (or an inner surface thereof) canalso be shaped to define at least a portion of the second fluid path 162of the biological sterilization indicator 100, for example, between anouter surface of the insert 130 and an inner surface of the housing 102.For example, in some embodiments, as shown in FIGS. 4-5, 8 and 9, thesidewall 131 of the insert 130 can include a channel (or groove, recess,or the like) 169 configured to form at least a portion of the secondfluid path 162.

The second fluid path 162 can function as an “internal vent” or a “ventchannel” within the biological sterilization indicator 100 to allow gas(e.g., displaced gas, such as air that had been trapped in the secondchamber 111 (e.g., near the closed end 105 of the biologicalsterilization indicator 100) to escape the second chamber 111 of thebiological sterilization indicator 100. In some embodiments, the secondfluid path 162 can provide an escape, or internal vent, for a gaspresent in the second chamber 111 during activation to facilitate movingthe liquid 122 into the second chamber 111 from the first chamber 109 asit is released from the container 120. Additionally or alternatively, insome embodiments, the second fluid path 162 can provide an escape, orinternal vent, for a gas present in the second chamber 111 duringsterilization to facilitate moving a sterilant into the second chamber111 of the biological sterilization indicator 100 and to the spores 115,with more efficient sterilant penetration into the second chamber 111.

By way of example only, as shown in FIGS. 5 and 10, the second fluidpath 162 can be at least partially defined by both a portion of theinsert 130 (e.g., the channel 169) and by a channel (or groove, recess,or the like) 163 formed in the wall 108 of the housing 102 (e.g., in aninner surface of the wall 108). However, it should be understood that insome embodiments, the second fluid path 162 can be formed entirely ofthe housing 102 or of various combinations of other components of thebiological sterilization indicator 100 such that the second fluid path162 provides fluid connection between the second chamber 111 and anotherinternal portion or region of the biological sterilization indicator100. For example, the second fluid path 162 need not be formed by boththe housing 102 and the insert 130, but can be formed by one of thesecomponents, or other components. In addition, as shown in FIGS. 5 and10, the channel 163 that defines at least a portion of the second fluidpath 162 is molded into an outer surface and an inner surface of thehousing 102, such that the channel 163 is visible on the inside and theoutside of the housing 102. However, the outer surface of the housing102 need not include such a shape, and rather, in some embodiments, theouter surface of the housing 102 can remain substantially uniform orunchanged, and the inner surface of the housing 102 (e.g., a wall 108 ofthe housing 102) can include the channel 163.

Furthermore, in some embodiments, neither the insert 130 nor the housing102 include the channel 169 or the channel 163, respectively, but ratherthe insert 130 and the housing 102 are shape and dimensioned such that aspace or gap is provided between the insert 130 and the housing 102 thatis in fluid communication with the second chamber 111, and such a spaceor gap functions as the second fluid path 162.

As further shown in FIGS. 7 and 9, in some embodiments, the first fluidpath 160 and/or the second fluid path 162 can be at least partiallydefined by one or more of the wall 118, the substrate 119, the insert130, and the housing 102. In addition, at least one of the first fluidpath 160 and the second fluid path 162 can be defined at least partiallyby the spore carrier 135, or a portion thereof.

In some embodiments, the biological sterilization indicator 100 caninclude the following components arranged in the following order whenthe container 120 is in a first, unfractured, state: the closed end 105of the housing 102 of the biological sterilization indicator 100, thesecond chamber 111, the substrate 119, the insert 130, the first chamber109, the container 120, the open end 101 of the housing 102 (or thesecond portion 106 of the housing 102).

As shown in the illustrated embodiment, the second fluid path 162 canallow the second chamber 111 to vent to another portion of thebiological sterilization indicator 100, such as the first chamber 109.In some embodiments, the second fluid path 162 can exit the secondchamber 111 at a position located above (e.g., vertically above) theposition at which the first fluid path 160 enters the second chamber111, particularly, in embodiments in which the second fluid path 162vents the second chamber 111 back to the first chamber 109. Said anotherway, in some embodiments, the second fluid path 162 can extend from thesecond chamber 111 to a position (e.g., a fourth level L₄, describedbelow) in the biological sterilization indicator 100 that is above theposition (e.g., a first level L₁ or a second level L₂, described below)at which the first fluid path 160 enters the second chamber 111.Furthermore, in some embodiments, the position at which the second fluidpath 162 enters the first chamber 109 can be located above (e.g.,vertically above) the position at which the first fluid path 160 entersthe second chamber 111.

In some embodiments, the first fluid path 160 can be positioned tofluidly couple the second chamber 111 with a proximal portion of thebiological sterilization indicator 100 (e.g., a portion of the firstchamber 109 that is located proximally or adjacent the second chamber111, e.g., at the first level L₁ and/or the second level L₂), and thesecond fluid path 162 can be positioned to fluidly couple the secondchamber 111 with a distal portion of the biological sterilizationindicator 100 (i.e., a portion of the first chamber 109 that is locatedfurther from the second chamber 111, e.g., at a third level L₃,described below, and/or the fourth level L₄). As a result, the positionat which the second fluid path 162 enters the first chamber 109 can bepositioned further from the second chamber 111 than the position atwhich the first fluid path 160 enters the second chamber 111.

More specifically and by way of example only, with reference to FIGS. 7and 9, in some embodiments, fluid can enter the second chamber 111 at avariety of locations, such as at the first level, height, or position(e.g., longitudinal position) L₁ located generally at the front of theinsert 130, the substrate 119, the housing 102, and/or the secondchamber 111, as well as at the second level, height, or position (e.g.,longitudinal position) L₂ located approximately at the level of theaperture 121 in the substrate 119. As described above, it should beunderstood that the variety of opening and spaces between the firstchamber 109 and the second chamber 111 that allow fluid to move into thesecond chamber 111 can collectively be referred to as the first fluidpath 160. As further illustrated in FIG. 7, in some embodiments, gas(e.g., displaced gas) can exit the second chamber 111 via the secondfluid path 162 (i.e., as fluid moves into the second chamber 111 via thefirst fluid path 160) at the third level, height, or position (e.g.,longitudinal position) L₃ located generally at the back of the insert130, the substrate 119, the housing 102, and/or the second chamber 111.

In the vertically upright orientation of the biological sterilizationindicator 100 shown in FIGS. 7 and 9, the third level L₃ is located ator above both the first level L₁ and the second level L₂. In addition,in some embodiments, the third level L₃ can still be located at or aboveboth the first level L₁ and the second level L₂ in operation of thebiological sterilization indicator 100 (e.g., when seated in a well of areading apparatus, during sterilization, and/or during activation). Thatis, in some embodiments, the biological sterilization indicator 100 canbe tilted in operation (e.g., toward the left-hand side of FIG. 7 or 9,toward the right-hand side of FIG. 4 or 6, into the page of FIG. 4 or 6,and/or out of the page of FIG. 7 or 9).

The first, second, and third levels L₁, L₂, and L₃ are shown by way ofexample only; however, it should be understood that the exact locationat which the first fluid path 160 enters the second chamber 111 and/orthe exact location at which the second fluid path 162 exits the secondchamber 111 can be different than what is illustrated in FIGS. 7 and 9.

As shown in FIGS. 7 and 9, the second fluid path 162 is at leastpartially defined by the channel 169 of the insert 130 and/or thechannel 163 of the housing 102, which will generally be referred to assimply “the channel” in the following discussion, which can beinterpreted to refer to at least a portion of the channel 163 and/or thechannel 169 of the illustrated embodiment. In the illustratedembodiment, the channel has an entrance that can be described as beinglocated at any point in the second chamber 111, or at the third levelL₃, and an exit that is positioned generally at the fourth level,height, or position (e.g., longitudinal position) L₄. As shown in FIGS.7 and 9, the exit position of the channel (i.e., the fourth level L₄) isgenerally located above the position at which the first fluid path 160connects with the second chamber 111 (i.e., the first level L₁ and/orthe second level L₂), for example, in operation of the biologicalsterilization indicator 100.

Said another way, the first fluid path 160 can be positioned to fluidlycouple the second (lower) end 113 of the first chamber 109 to the first(upper) end 124 of the second chamber 111. The second fluid path 162, onthe other hand, can be positioned to fluidly couple the second chamber111 (e.g., the first (upper) end 124 of the second chamber 111) to anupper portion (e.g., the first (upper) end 112) of the first chamber109.

Furthermore, in some embodiments, the position or level at which thesecond fluid path 162 (or the channel) connects with the second chamber111 can be described as being located at portion of the second chamber111 that is the last to fill with the liquid 122 when the container 120is in its second, fractured, state.

In some embodiments, when the container 120 is in the second, fractured,state, and the second chamber 111 is at least partially filled with theliquid 122, the liquid 122 can have a level, height or position (e.g.,longitudinal position) L, and the second fluid path 162 can extendbetween a position below the level L and a position above the level L.As a result, as the second chamber 111 fills with the liquid 122 whenthe container is in the second state, the second chamber 111 cancontinually be vented by the second fluid path 162.

In some embodiments, the first fluid path 160 can function as the mainor primary fluid communication path between the first chamber 109 andthe second chamber 111, and the second fluid path 162 can serve as anaccessory or secondary fluid communication path between the secondchamber 111 and the first chamber 109 (e.g., when the second fluid path162 exits in the first chamber 109 and not another portion of thebiological sterilization indicator 100). In such embodiments, thecollective space, volume and/or area of the second fluid path 162 can besubstantially less than that of the first fluid path 160. In someembodiments, at least a portion of the first fluid path 160 and thesecond fluid path 162 can be described as being substantially isolatedfrom one another or as being substantially parallel andnon-intersecting. In some embodiments, the first fluid path 160 and thesecond fluid path 162 can each extend substantially longitudinally(e.g., substantially parallel to the longitudinal direction D_(L))between the first chamber 109 and the second chamber 111.

That is, generally, the biological sterilization indicator 100 thatincludes (1) a first fluid path, such as the first fluid path 160,configured to accommodate at least a majority of the fluid movement fromthe first chamber 109 to the second chamber 111, and (2) a second fluidpath, such as the second fluid path 162, configured to vent gas from thesecond chamber 111 would have advantages over a biological sterilizationindicator 100 that included either only one internal chamber, or onlyone fluid path connecting the first chamber 109 and the second chamber111, such that gas would have to exit the second chamber 111 via thesame fluid path that fluid enters the second chamber 111.

By configuring the first fluid path 160 and the second fluid path 162 asshown in the illustrated embodiment, in some embodiments, the biologicalsterilization indicator 100 can at least partially eliminate anyair-lock effect that may occur as a result of trying to move a sterilantand/or the liquid 122 into the second chamber 111. In addition, in someembodiments, the second fluid path 162 can allow for the biologicalsterilization indicator 100 to be activated, and the liquid 122 to bemoved into the second chamber 111 due to gravity, while the biologicalsterilization indicator 100 remains in the same orientation (e.g., asubstantially vertically upright orientation, as shown in FIGS. 4-5, 7and 9), without requiring that the biological sterilization indicator100 to be tipped upside down, or otherwise re-oriented in order to movethe liquid 122 into the second chamber 111.

With continued reference to the insert 130, the projections 158 of theinsert 130 are illustrated as being relatively rigid and stationary.That is, in some embodiments, the projections 158 may not be adapted tosubstantially flex, distort, deform or otherwise heed to the container120 as it is moved in the housing 102. Rather, in some embodiments, asshown in FIGS. 4-7 and 9, the projections 158 can each be configured tohave an upper end 159 atop which the container 120 can be positioned andheld intact before activation. As shown in FIGS. 4-5 and 7, in someembodiments, the projections 158 can be positioned to fracture thecontainer 120 at its radiused end, for example, when an oblong orcapsule-shaped container 120 is employed.

One potential advantage of having the projections 158 form at least aportion of the carrier 132 is that the bottom of the container 120 canbe unrestricted when the container 120 is fractured, such that theliquid 122 can be released from the container 120 and moved toward thespores 115 with relative ease and reliability.

In such embodiments, the insert 130 can be used to fracture thecontainer 120 in a direction that is substantially perpendicular to aflat side of the container 120, for example, when an oblong orcapsule-shaped container 120 is employed. In such embodiments,fracturing the container 120 along its side can be achieved, along withmaintaining some open spaces around the lower end of the container 120to facilitate moving the liquid 122 from the container 120 to theproximity of the spores 115 when the container 120 is fractured.

As mentioned above, the projections 158 can be adapted to fracture thecontainer 120 as the container 120 is moved with respect to the housing102 (e.g., along the longitudinal direction D_(L)), for example, inresponse to the second portion 106 of the housing 102 being moved withrespect to the first portion 104 of the housing 102 (e.g., from thefirst position 148 to the second position 150).

In some embodiments, the projections 158 can include one or more edges(e.g., tapered edges) or points or otherwise be configured toconcentrate the crushing force to increase the pressure on the container120 in the regions adjacent the projections 158, and to facilitatefracturing the container 120 more easily and in one or more desiredregions. In some embodiments, such concentration of force can reduce thetotal effort or force needed to move the second portion 106 with respectto the first portion 104 and to fracture the container 120 (or a portionthereof).

As shown in FIGS. 4-7 and 9, the projections 158 are integrally formedwith the base 127 of the insert 130; however, it should be understoodthat the projections 158 can instead be integrally formed with the wall108 of the housing 102. In addition, in some embodiments, theprojections 158 can be coupled to the housing 102, or the projections158 and the base 127 can be provided by separate inserts. In suchembodiments, the projections 158 can each be a separate insert, ormultiple projections 158 can be provided by one or more inserts. Inaddition, the insert 130 can be configured to abut the wall 118 toinhibit movement of the first portion the insert 130 into the proximityof the spores 115 (e.g., the lower portion 114 of the housing 102).

In addition, in some embodiments, as shown in FIGS. 4-7 and 9, theprojections 158 can extend a distance along the longitudinal directionD_(L), and the length and/or thickness (e.g., which can vary along thelength) of the projections 158 can be tailored to control the fracturingof the container 120 at a desired position in the housing 102 and in adesired manner. The configuration of the projections 158 is shown inFIGS. 4-10 by way of example only.

In general, each of the projections 158 is shown by way of example onlyas increasing in thickness (e.g., inwardly toward the container 120 orcenter of the housing 102) along the longitudinal direction D_(L) towardthe spores 115. Such a configuration can decrease the cross-sectionalarea that is available to the container 120, as the container 120 ismoved toward the spores 115, for example, in response to the secondportion 106 being moved to the second position 150.

Furthermore, the biological sterilization indicator 100 is shown inFIGS. 3-10 as including two projections 158 and a sidewall 131 by way ofexample only, but it should understood that one projection 158 or asmany as structurally possible, and other configurations, can beemployed. In addition, the projections 158 can be shaped and dimensionedas desired, depending on the shape and dimensions of the housing 102, onthe shape and dimensions of the container 120, on the shape anddimensions of the insert 130, and/or on the manner and position desiredfor fracturing the container 120.

As mentioned above, in some embodiments, at least a portion of thehousing 102 can be tapered (see, e.g., the tapered portion 146 in FIG.6). As a result, the cross-sectional area in the housing 102 cangenerally decrease along the longitudinal direction D_(L). However, itshould be understood that the inner dimensions of the housing 102 cangenerally decrease in the tapered portion along the longitudinaldirection D₁ without the outer dimensions of the housing 102 changing.In some embodiments, the outer dimensions of the housing 102 can beuniform along its length, even though the inner portion of the housing102 tapers along its length. In some embodiments, the one or moreprojections 158 alone can vary in thickness (i.e., toward the container120, e.g., in a radial direction) along the longitudinal directionD_(L), such that the cross-sectional area available to the container 120generally decreases as the container 120 is moved in the housing 102during activation, even though the dimensions of the housing 102 do notchange (e.g., even if the housing 102 does not include any taperedportion 146, either internally or externally).

As shown in FIGS. 4-10, the upper end 159 of each of the projections 158includes a rounded, curved or arcuate surface, which can facilitatemovement of the container 120 from the first position 148 in which thecontainer 120 sits at least partially above the upper end 159 of theprojection 158 to a position in which the container 120 is forced, atleast partially, into the smaller cross-sectional area region in betweenthe projections 158 (or between the wall 108 of the housing 102 and oneor more projections 158). In addition, the rounded upper end 159 caninhibit premature breakage of the container 120, which can inhibitpremature activation of the biological sterilization indicator 100(i.e., premature release of the liquid 122).

In some embodiments, as shown in FIG. 6, the insert 130 can be sized andshaped to allow the container 120 to be held above the projections 158and out from the region adjacent any portion of an inwardly-facingsurface of one or more of the projections 158 to inhibit accidental orpremature activation of the biological sterilization indicator 100. Sucha configuration can also inhibit inadvertent breakage due to shock ormaterial expansion (e.g., due to exposure to heat during a sterilizationprocess).

The carrier 132, which can be formed at least partially by the upperends 159 of the projections 158, can be configured to hold a bottomportion of the container 120, and the projections 158 can be positionedto fracture the container 120 at a location near the bottom of thecontainer 120 as it is positioned in the housing 102. Such aconfiguration can allow the container 120 to be broken near its bottomand can facilitate removal of the liquid 122 from the container 120,which can enhance the availability of the liquid 122 to the spores 115,and can enhance the reliability of releasing the liquid 122 into fluidcommunication with the spores 115 (e.g., with the spore reservoir 136).Such a configuration is shown by way of example only, however, and itshould be understood that the projections 158 can be configured andpositioned to fracture the container 120 in any desired manner.

Some embodiments of the present disclosure provide optimal and safebreakage of a frangible container 120 with relatively low force, whileenhancing transfer of liquid 122 to the spore region (e.g., the secondchamber 111 of the housing 102) of the biological sterilizationindicator 100, and/or enhancing containment of the liquid 122 in thespore region of the biological sterilization indicator 100. In addition,some embodiments of the present disclosure operate to drive a liquid toa particular area of the biological sterilization indicator 100, such asa detection chamber (e.g., the second chamber 111) of the biologicalsterilization indicator 100.

In the embodiment illustrated in FIGS. 4-10, the insert 130 isillustrated as including two projections 158 that are approximatelyequally spaced about the container 120 and/or about the sidewall 131.However, in some embodiments, the sidewall 131 can include one solid(e.g., substantially annular or semi-annular) projection 158 thatextends radially inwardly from the sidewall 131. Furthermore, in someembodiments, the sidewall 131 can extend further around the innersurface of the housing 102 than what is illustrated. However, employingone or more narrower (e.g., in an angular dimension) projections 158,such as those shown in FIGS. 4-10, can provide a substantially constantor substantially unobstructed sterilant path 164 around the container120.

Whether the insert 130 includes one or more projections 158 or sidewalls131, the insert 130 can be configured to hold the container 120 in thehousing 102 in a consistent location to provide a substantially constantsterilant path 164 during sterilization. For example, rather thanallowing the container 120 to move or roll around (e.g., radially and/orlongitudinally) in the housing 102 before activation (e.g., duringsterilization), the insert 130 can hold the container 120 in asubstantially consistent position, which can allow a sterilant asubstantially consistent and relatively unobstructed path between anouter surface of the container 120 and an inner surface of the housing102, with little or no opportunity for inadvertent blockage.

As shown in the illustrated embodiment, the insert 130 can furtherinclude one or more projections 161 positioned substantiallyhorizontally or perpendicularly with respect to the longitudinaldirection D_(L) of a biological sterilization indicator (e.g., when theinsert 130 is positioned in a biological sterilization indicator). Theprojections 161 can be referred to as “second projections” or“horizontal projections,” while the projections 158 used to hold and/orbreak the container 120 can be referred to as “first projections” or“vertical projections.” The second projections 161 are not angleddownwardly like the base 127. As a result, the second projections 161can be used for a variety of purposes. For example, the secondprojections 161 can stabilize the insert 130 (e.g., aid in holding theinsert 130 in a desired position in the housing 102 of the biologicalsterilization indicator 100) under the force of fracturing the container120. In addition, the second projections 161 can function to retainand/or collect fractured portions of the container 120 after it has beenfractured to inhibit movement of such portions into the proximity ofspores in the biological sterilization indicator, which could negativelyaffect spore growth and/or detection of spore growth. Other shapes andconfigurations of the second projections 161 can be employed that stillallow for fluid movement down to the spores 115 while inhibiting solidmovement down to the spores 115.

In some embodiments, the insert 130 (e.g., the base 127) can be adaptedfor one or more of facilitating or allowing fluid movement (e.g.,movement of the liquid 122) into the second chamber 111 (i.e., the lowerportion 114) of the housing 102; minimizing movement of fractions orportions (e.g., solids) of the fractured container 120 into the secondchamber 111 of the housing 102, that is, collecting and/or retainingportions of the fractured container 120; and/or minimizing diffusion ofthe spores 115 and/or signals out of the second chamber 111 of thehousing 102. For example, in some embodiments, the base 127 can beconfigured to function as a grate or filter. In some embodiments, sporegrowth is determined by fluorescent indicators/molecules (e.g.,fluorophores) or other markers. In some embodiments, if the liquid levelafter activation in the biological sterilization indicator 100 is abovethe location of the spores 115, such molecules or markers, or the spores115 themselves, can move or diffuse away from or out of the sporereservoir 136 and, potentially, out of the second chamber 111 of thehousing 102. As a result, portions of the biological sterilizationindicator 100 (e.g., the insert 130) can be configured to inhibitundesirable diffusion of various indicators, molecules, and/or markersout of the second chamber 111 of the biological sterilization indicator100. In some embodiments, as described above, the substrate 119 can alsoinhibit such undesirable diffusion.

In the embodiment illustrated in FIGS. 4-7, the base 127 of the insert130 is generally U-shaped or horseshoe-shaped and includes a centralaperture 177 (see FIG. 5) that facilitates the movement of sterilanttoward the spores 115 during sterilization and the movement of theliquid 122 toward the spores 115 during activation. The horseshoe shapeof the base 127 can increase the opening between the upper portion 116(i.e., the first chamber 109) and the lower portion 114 (i.e., thesecond chamber 111) of the housing 102; however, this shape is shown byway of example only, and other shapes can be employed.

In some embodiments, the insert 130 can be described as including one ormore downwardly-extending projections 127 adapted to abut or otherwisecouple to the wall 118 or another internal structure of the biologicalsterilization indicator 100 to provide a base or support for the insert130, to inhibit movement of the insert 130 and container 120 relative tothe housing 102 before activation, and/or to provide resistance or forceto aid in breaking the container 120 during activation. As a result, insome embodiments, the base 127 can instead be referred to as “thirdprojections” 127.

As shown in the illustrated embodiment, in some embodiments, the insert130 can be configured to reside entirely in the first chamber 109 of thebiological sterilization indicator 100, such that the insert 130 doesnot extend into the second chamber 111 where it could potentiallyinterfere with interrogation or detection processes. Furthermore, theinsert 130 can be configured to inhibit movement of other portions ofthe biological sterilization indicator 100 (e.g., the fracturedcontainer 120) into the second chamber 111.

The insert 130 of the illustrated embodiment is generally symmetricalabout a central longitudinal line of symmetry, such that there are twoidentical first projections 158, two identical second projections 161,and two identical third projections 127. However, the insert 130 neednot include any lines of symmetry, and the first projections 158 neednot be the same as one another, the second projections 161 need not bethe same as one another, and the third projections 127 need not be thesame as one another. The insert 130, and the various projections 158,161 and 127 can be sized and positioned to control the sterilant path164, for example, to tailor the kill/survival rate of the biologicalsterilization indicator 100, to inhibit inadvertent fracture of thecontainer 120, to facilitate movement of the container 120 in thehousing 120, to mate with or engage the housing 102, and/or to controlthe breakage of the container 120.

By way of example only, the illustrated insert 130 is shown as being aunitary device that includes at least the following: means for holdingthe container 120 before activation, for fracturing the container 120during activation; for allowing movement of the container 120 in thehousing 102; for providing a substantially constant sterilant path 164,for collecting and/or retaining portions of the fractured container 120after activation (or at least partially inhibiting movement of portionsof the fractured container 120 into the second chamber 111 of thehousing 102); and/or for minimizing diffusion of the spores 115 and/orsignals from the second chamber 111 to the first chamber 109 of thehousing 102 after activation. However, it should be understood that insome embodiments, the insert 130 can include multiple portions that maynot be part of a single, unitary device, and each of the portions can beadapted to do one or more of the above functions.

The insert 130 is referred to as an “insert” because in the illustratedembodiment, the device that performs the above functions is a devicethat can be inserted into the reservoir 103 (and, particularly, thefirst chamber 109) of the housing 102. However, it should be understoodthat the insert 130 can instead be provided by the housing 102 itself oranother component of the biological sterilization indicator 100 and neednot necessarily be insertable into the housing 102. The term “insert”will be described throughout the present disclosure for simplicity, butit should be understood that such a term is not intended to be limiting,and it should be appreciated that other equivalent structures thatperform one or more of the above functions can be used instead of, or incombination with, the insertable insert 130. Furthermore, in theillustrated embodiment, the insert 130 is both insertable into andremovable from the housing 102, and particularly, into and out of thefirst portion 104 (and the first chamber 109) of the housing 102.However, it should be understood that even if the insert 130 isinsertable into the housing 102, the insert 130 need not be removablefrom the housing 102, but rather can be fixedly coupled to the housing102 in a manner that inhibits removal of the insert 130 from the housing102 after positioning the insert 130 in a desired location.

In some embodiments, at least a portion of the housing 102, for example,the lower portion 114 of the housing 102, can be transparent to anelectromagnetic radiation wavelength or range of wavelengths (e.g.,transparent to visible light when visible-light optical detectionmethods are employed), which can facilitate detection of spore growth.That is, in some embodiments, as shown in FIGS. 6, 7 and 9, at least aportion of the housing 102 can include or form a detection window 167.

In addition, in some embodiments, as shown in FIG. 6, at least a portionof the housing 102, for example, the lower portion 114 can include oneor more planar walls 168. Such planar walls 168 can facilitate detection(e.g., optical detection) of spore growth. In addition, as shown anddescribed above, the wall 108 of the first portion 104 of the housing102 can include one or more stepped or tapered regions, such as the step152, the step 123, and a tapered wall, or step, 170. The tapered wall170 can function to reduce the overall thickness and size of the lowerportion, or detection portion, 114 of the housing 102, such that theouter dimensions of the housing 102 are reduced in addition to the innerdimensions. Such a reduction in size and/or thickness of the lowerportion 114 of the biological sterilization indicator 100 can facilitatedetection. In addition, having one or more features, such as the stepsand/or tapered walls 123, 152, 170 can allow the biologicalsterilization indicator 100 to be coupled to a reader or detectiondevice in only one orientation, such that the biological sterilizationindicator 100 is “keyed” with respect to a reading apparatus, which canminimize user error and enhance reliability of a detection process. Insome embodiments, one or more portions of the biological sterilizationindicator 100 can be keyed with respect to a reading apparatus.

The biological sterilization indicator of the present disclosuregenerally keeps the liquid 122 and the spores 115 separate but inrelatively close proximity (e.g., within the self-contained biologicalsterilization indicator 100) during sterilization, such that the liquid122 and the spores 115 can be readily combined after exposure to asterilization process. The liquid 122 and the spores 115 can beincubated during a detection process (e.g., the reading apparatus 12 canincubate the biological sterilization indicator 100), or the biologicalsterilization indicator 100 can be incubated prior to a detectionprocess. In some embodiments, when incubating the spores with the liquid122, an incubation temperature above room temperature can be used. Forexample, in some embodiments, the incubation temperature is at leastabout 37° C., in some embodiments, the incubation temperature is atleast about 50° C. (e.g., 56° C.), and in some embodiments, at leastabout 60° C. In some embodiments, the incubation temperature is nogreater than about 60° C., in some embodiments, no greater than about50° C., and in some embodiments, no greater than about 40° C.

A detection process can be adapted to detect a detectable change fromthe spores 115 (e.g., from within the spore reservoir 136) or the liquid122 surrounding the spores 115. That is, a detection process can beadapted to detect a variety of characteristics, including, but notlimited to, electromagnetic radiation (e.g., in the ultraviolet,visible, and/or infrared bands), fluorescence, luminescence, lightscattering, electronic properties (e.g., conductance, impedance, or thelike, or combinations thereof), turbidity, absorption, Ramanspectroscopy, ellipsometry, or the like, or a combination thereof.Detection of such characteristics can be carried out by one or more of afluorometer, a spectrophotometer, colorimeter, or the like, orcombinations thereof. In some embodiments, such as embodiments thatmeasure fluorescence, visible light, etc., the detectable change ismeasured by detecting at a particular wavelength.

The spores and/or the liquid 122 can be adapted (e.g., labeled) toproduce one or more of the above characteristics as a result of abiochemical reaction that is a sign of spore viability. As a result, nodetectable change (e.g., as compared to a baseline or backgroundreading) can signify an effective sterilization process, whereas adetectable change can signify an ineffective sterilization process. Insome embodiments, the detectable change can include a rate at which oneor more of the above characteristics is changing (e.g., increasingfluorescence, decreasing turbidity, etc.).

In some embodiments, spore viability can be determined by exploitingenzyme activity. As described in Matner et al., U.S. Pat. No. 5,073,488,entitled “Rapid Method for Determining Efficacy of a Sterilization Cycleand Rapid Read-out Biological Indicator,” which is incorporated hereinby reference, enzymes can be identified for a particular type of sporein which the enzyme has particularly useful characteristics that can beexploited to determine the efficacy of a sterilization process. Suchcharacteristics can include the following: (1) the enzyme, whensubjected to sterilization conditions which would be sufficient todecrease a population of 1×10⁶ test microorganisms by about 6 logs(i.e., to a population of about zero as measured by lack of outgrowth ofthe test microorganisms), has a residual activity which is equal to“background” as measured by reaction with a substrate system for theenzyme; and (2) the enzyme, when subjected to sterilization conditionssufficient only to decrease the population of 1×10⁶ test microorganismsby at least 1 log, but less than 6 logs, has enzyme activity greaterthan “background” as measured by reaction with the enzyme substratesystem. The enzyme substrate system can include a substance, or mixtureof substances, which is acted upon by the enzyme to produce a detectableenzyme-modified product, as evident by a detectable change.

In some embodiments, the biological sterilization indicator 100 can beassayed in a single-side mode, where the biological sterilizationindicator 100 includes only one detection window (e.g., detection window167 of FIG. 6) that is positioned, for example, near the spores 115. Insome embodiments, however, the biological sterilization indicator 100can include more than one detection window (e.g., a window formed by allor a portion of both parallel walls 168 of the lower portion 114 of thehousing 102), such that the biological sterilization indicator 100 canbe assayed via more than one detection window. In embodiments employingmultiple detection windows, the detection windows can be positionedside-by-side (similar to a single-side mode), or the detection windowscan be oriented at an angle (e.g., 90 degrees, 180 degrees, etc.) withrespect to one another.

In general, the spores 115 are positioned within the spore reservoir 136which is in fluid communication with the reservoir 103. In someembodiments, the spore reservoir 136 forms a portion of the reservoir103 (e.g., a portion of the second chamber 111). As shown in FIG. 7, thereservoir 103 is in fluid communication with ambience (e.g., via theaperture 107) during sterilization to allow sterilant to enter thereservoir 103 during a sterilization process to sterilize the spores115. The container 120 can be configured to contain the liquid 122during sterilization to inhibit the liquid 122 from being in fluidcommunication with the spores 115, the reservoir 103, and the sterilantduring sterilization.

Various details of the spores 115 and/or spore reservoir 136 will now bedescribed in greater detail.

In some embodiments, the spores 115 can be positioned directly in thelower portion 114 of the housing 102, or the spores 115 can bepositioned in a spore reservoir, such as the spore reservoir 136 (e.g.,provided by the spore carrier 135). Whether the spores 115 arepositioned directly in the lower portion 114 of the housing 102 or in aspore reservoir, the spores 115 can be provided in a variety of ways. Insome embodiments, the spores 115 can be in a spore suspension that canbe positioned in a desired location in the biological sterilizationindicator 100 and dried down. In some embodiments, the spores 115 can beprovided on a substrate (not shown) that can be positioned and/orsecured in a desired location in the biological sterilization indicator100. Some embodiments can include a combination of spores 115 providedin a dried down form and spores 115 provided on a substrate.

In some embodiments, the substrate can be positioned to support thespores 115 and/or to help maintain the spores 115 in a desired locus.Such a substrate can include a variety of materials, including, but notlimited to, paper, a polymer (e.g., any of the polymers listed abovewith respect to the housing 102), an adhesive (e.g., acrylate, naturalor synthetic rubber, silicone, silicone polyurea, isocyanate, epoxy, orcombinations thereof), a woven cloth, a nonwoven cloth, a microporousmaterial (e.g., a microporous polymeric material), a reflective material(e.g., a metal foil), a glass, a porcelain, a ceramic, a gel-formingmaterial (e.g., guar gum), or combinations thereof. In addition, oralternatively, such a substrate can include or be coupled to ahydrophilic coating to facilitate bringing the liquid 122 into intimatecontact with the spores 115 (e.g., when the liquid 122 employed isaqueous). In addition, or alternatively, such a hydrophilic coating canbe applied to any fluid path positioned to fluidly couple the liquid 122and the spores 115. In some embodiments, in addition to, or in lieu of ahydrophilic coating, a hydrophobic coating can be applied to otherportions of the housing 102 (e.g., the lower portion 114 of the housing102) and/or spore reservoir 136, such that the liquid 122 ispreferentially moved into contact with the spores 115.

Some embodiments of the biological sterilization indicator 100 do notinclude the spore carrier 135. Rather, the spore reservoir 136 isprovided by the lower portion 114 of the housing 102 itself, and thespores 115 can be positioned in the lower portion 114, adsorbed to aninner surface or wall of the lower portion 114, or combinations thereof.In some embodiments, the spores 115 can be provided on a substrate thatis positioned in the lower portion 114 of the housing 102.

In some embodiments, the spores 115 can be positioned in one locus ofspores or in a plurality of loci of spores, all of which can bepositioned either in the reservoir 103, in the lower portion 114 of thehousing 102, and/or in the spore reservoir 136. In some embodiments,having multiple loci of spores can maximize the exposure of the sporesto sterilant and to the liquid 122, can improve manufacturing (e.g.,placement of the spores can be facilitated by placing each locus ofspores in a depression within the biological sterilization indicator100), and can improve detection characteristics (e.g., because spores inthe middle of one large locus of spores may not be as easily detected).In embodiments employing a plurality of loci of spores, each locus ofspores can include a different, known number of spores, and/or eachlocus of spores can include different spores, such that a plurality ofspore types can be tested. By employing multiple types of spores, thebiological sterilization indicator 100 can be used for a variety ofsterilization processes and a specific locus of spores can be analyzedfor a specific sterilization process, or the multiple types of sporescan be used to further test the effectiveness, or confidence, of asterilization process.

In addition, in some embodiments, the biological sterilization indicator100 can include a plurality of spore reservoirs 136, and each sporereservoir 136 can include one or more loci of spores 115. In someembodiments employing a plurality of spore reservoirs 136, the pluralityof spore reservoirs 136 can be positioned in fluid communication withthe reservoir 103.

In some embodiments, the spores 115 can be covered with a cover (notshown) adapted to fit in or over the spores 115 and/or the sporereservoir 136. Such a cover can help maintain the spores within thedesired region of the biological sterilization indicator 100 duringmanufacturing, sterilization and/or use. The cover, if employed, can beformed of a material that does not substantially impede a detectionprocess, and/or which is at least partially transmissive toelectromagnetic radiation wavelengths of interest. In addition,depending on the material makeup of the cover, in some embodiments, thecover can facilitate wicking the liquid 122 (e.g., the nutrient medium)along the spores 115. In some embodiments, the cover can also containfeatures for facilitating fluid flow into the spore reservoir 136 (or tothe spores 115), such as capillary channels, hydrophilic microporousfibers or membranes, or the like, or a combination thereof. In addition,in some embodiments, the cover can isolate a signal, or enhance thesignal, which can facilitate detection. Such a cover can be employedwhether the spores 115 are positioned within the spore reservoir 136 ordirectly in the lower portion 114 of the housing 102. In addition, sucha cover can be employed in embodiments employing a plurality of loci ofspores. The cover can include a variety of materials, including, but notlimited to, paper, a polymer (e.g., any of the polymers listed abovewith respect to the housing 102), an adhesive (e.g., acrylate, naturalor synthetic rubber, silicone, silicone polyurea, isocyanate, epoxy, orcombinations thereof), a woven cloth, a nonwoven cloth, a microporousmaterial (e.g., a microporous polymeric material), a glass, a porcelain,a ceramic, a gel-forming material (e.g., guar gum), or combinationsthereof.

In some embodiments, the biological sterilization indicator 100 canfurther include a modified inner surface, such as a reflective surface,a white surface, a black surface, or another surface modificationsuitable to optimize the optical properties of the surface. A reflectivesurface (e.g., provided by a metal foil) can be positioned to reflect asignal sent into the spore reservoir 136 from an assaying or detectiondevice and/or to reflect any signal generated within the spore reservoir136 back toward the assaying device. As a result, the reflective surfacecan function to improve (e.g., improve the intensity of) a signal fromthe biological sterilization indicator 100. Such a reflective surfacecan be provided by an inner surface of the housing 102; a materialcoupled to the inner surface of the housing 102; an inner surface thespore reservoir 136; a material coupled to the inner surface of thespore reservoir 136; or the like; or the reflective surface can form aportion of or be coupled to a spore substrate; or a combination thereof.

Similarly, in some embodiments, the biological sterilization indicator100 can further include a white and/or black surface positioned toincrease and/or decrease a particular signal sent into the sporereservoir 136 from an assaying device and/or to increase and/or decreasea particular signal generated within the spore reservoir 136. By way ofexample only, a white surface can be used to enhance a signal, and ablack surface can be used to reduce a signal (e.g., noise).

In some embodiments, the spores 115 can be positioned on afunctionalized surface to promote the immobilization of the spores 115on the desired surface. For example, such a functionalized surface canbe provided by an inner surface of the housing 102, an inner surface ofthe spore reservoir 136, can form a portion of or be coupled to a sporesubstrate, or the like, or a combination thereof.

In some embodiments, the spores 115 are positioned (e.g. applied bycoating or another application method) on a microstructured ormicroreplicated surface (e.g., such microstructured surfaces as thosedisclosed in Halverson et al., International Publication No. WO2007/070310, Hanschen et al., US. Publication No. US 2003/0235677, andGraham et al., International Publication No. WO 2004/000569, all ofwhich are incorporated herein by reference). For example, such amicrostructured surface can be provided by an inner surface of thehousing 102, can be provided by an inner surface of the spore reservoir136, can form a portion of or be coupled to a spore substrate, or thelike, or a combination thereof.

In some embodiments, the biological sterilization indicator 100 canfurther include a gel-forming material positioned to be combined withthe spores 115 and the liquid 122 when the liquid 122 is released fromthe container 120. For example, the gel-forming material can bepositioned near the spores 115 (e.g., in the spore reservoir 136), inthe lower portion 114 of the housing 102, can form a portion of or becoupled to a spore substrate, or the like, or a combination thereof.Such a gel-forming material can form a gel (e.g., a hydrogel) or amatrix comprising the spores and nutrients when the liquid 122 comesinto contact with the spores. A gel-forming material (e.g., guar gum)can be particularly useful because it has the ability to form a gel uponhydration, it can aid in localizing a signal (e.g., fluorescence), itcan anchor the spores 115 in place, it can help minimize diffusion ofthe spores 115 and/or a signal from the spore reservoir 136, and/or itcan enhance detection.

In some embodiments, the biological sterilization indicator 100 canfurther include an absorbent or a wicking material. For example, thewicking material can be positioned near the spores 115 (e.g., in thespore reservoir 136), can form at least a portion of or be coupled to aspore substrate, or the like, or a combination thereof. Such a wickingmaterial can include a porous wicking pad, a soaking pad, or the like,or a combination thereof, to facilitate bringing the liquid 122 intointimate contact with the spores.

In some embodiments, the frangible container 120 can be configured tofacilitate fracturing of the frangible container 120 in a desiredmanner. For example, in some embodiments, a lower portion of thefrangible container 120 can be formed of a thinner and/or weakermaterial, such that the lower portion preferentially fractures overanother portion of the frangible container 120. In addition, in someembodiments, the frangible container 120 can include a variety offeatures positioned to facilitate fracturing of the frangible container120 in a desired manner, including, but not limited to, a thin and/orweakened area, a score line, a perforation, or the like, or combinationsthereof.

The frangible container 120 can have a first closed state in which theliquid 122 is contained within the frangible container 120 and a secondopen state in which the frangible container 120 has fractured and theliquid 122 is released into the reservoir 103 and/or the spore reservoir136, and in fluid communication with the spores 115.

In some embodiments, the biological sterilization indicator 100 can beactivated (e.g., the second portion 106 can be moved to the secondposition 150) manually. In some embodiments, the biologicalsterilization indicator 100 can be activated by a reading apparatus(e.g., as the biological sterilization indicator 100 is positioned inthe reading apparatus). In some embodiments, the biologicalsterilization indicator 100 can be activated with a device (e.g., anactivation device) independent of such a reading apparatus, for example,by positioning the biological sterilization indicator 100 in the deviceprior to positioning the biological sterilization indicator 100 in awell of a reading apparatus. In some embodiments, the biologicalsterilization indicator 100 can be activated by a combination of two ormore of the reading apparatus, a device independent of the readingapparatus, and manual activation.

One or both of the biological sterilization indicator 100 and anotherdevice, such as a reading apparatus can be further configured to inhibitpremature or accidental fracturing of the frangible container 120. Forexample, in some embodiments, the biological sterilization indicator100, activation device, or reading apparatus can include a lock orlocking mechanism that is positioned to inhibit the second portion 106of the housing 102 from moving into the second position 150 untildesired. In such embodiments, the biological sterilization indicator 100cannot be activated until the lock is moved, removed or unlocked. Inaddition, or alternatively, in some embodiments, the biologicalsterilization indicator 100, activation device, and/or reading apparatuscan include a lock or locking mechanism that is positioned to inhibitthe second portion 106 of the housing 102 from moving from the secondposition 150 back into the first position 148 after activation.

In some embodiments, as shown in the illustrated embodiment, at least aportion of the housing can be flat (e.g., the parallel walls 168), andcan be substantially planar with respect to the spore reservoir 136, andone or both of the parallel walls 168 or a portion thereof (e.g., thedetection window 167) can be sized such that at least one dimension ofthe wall 168 (or detection window 167) substantially matches at leastone dimension of the spore reservoir 136 and/or the locus of spores 115.Said another way, the wall 168 or a portion thereof (e.g., the detectionwindow 167) can include a cross-sectional area that is substantially thesame size as the cross-sectional area of the spore reservoir 136 and/orthe locus of spores 115. Such size matching between the wall168/detection window 167 and the spore reservoir 136 and/or the locus ofspores 115 can maximize the signal detected during a detection orassaying process. Alternatively, or in addition, the wall 168 ordetection window 167 can be sized to match the reservoir 103 (e.g., atleast one dimension or the cross-sectional areas can be sized to match).Such size matching between detection zones can improve spore assayingand detection.

The biological sterilization indicator 100 illustrated in FIGS. 4-10, atleast the portion of the biological sterilization indicator 100 wherethe spores 115 are positioned, is relatively thin (i.e., the “zdimension” is minimized), such that an optical path from the spores tothe wall 168 (or detection window 167) is minimized and/or any effect ofinterfering substances in the liquid 122 (or nutrient medium) isminimized.

In use, the biological sterilization indicator 100 can be placed alongwith a sterilizing batch for a sterilization process. Duringsterilization, a sterilant is in fluid communication with the reservoir103 (i.e., the first chamber 109 and the second chamber 111), the sporereservoir 136, and the spores 115 primarily via the sterilant path 164,such that sterilant can reach the spores to produce sterilized spores.As described above, the cooperation of the first fluid path 160 and thesecond fluid path 162 can facilitate movement of the sterilant into thesecond chamber 111, and particularly, into the closed end 105 of thebiological sterilization indicator 100. In addition, duringsterilization, the frangible container 120 is in a closed state, heldintact at least partially by the carrier 132 of the insert 130. When thefrangible container 120 is in a closed state, the liquid 122 isprotected from the sterilant and is not in fluid communication with thereservoir 103 (particularly, the second reservoir 111 formed at leastpartially by the lower portion 114 of the housing 102), the sporereservoir 136, the spores 115, or the sterilant path 164.

Sterilization can further include moving a sterilant from the firstchamber 109 to the second chamber 111 via the first fluid path 160 whenthe container 120 is in the first state, and moving displaced gas (e.g.,trapped air) out of the second chamber 111 via the second fluid path 162in response to, or to facilitate, moving the sterilant from the firstchamber 109 to the second chamber 111.

Following sterilization, the effectiveness of the sterilization processcan be determined using the biological sterilization indicator 100. Thesecond portion 106 of the housing 102 can be unlocked, if previouslylocked in the first position 148, and moved from the first position 148(see FIG. 6) to the second position 150 (see FIG. 7) to cause activationof the biological sterilization indicator 100. Such movement of thesecond portion 106 can cause the frangible container 120 to move in thehousing 102, for example, along the longitudinal direction D_(L) from aposition above the upper ends 159 of the projections 158 to a positionwithin the interior of the projections 158, which can cause thefrangible container 120 to fracture. Fracturing the frangible container120 can change the frangible container 120 from its closed state to itsopen state and release the liquid 122 into the reservoir 103, and intofluid communication with the spore reservoir 136 and the spores 115. Theliquid 122 can either include nutrient medium (e.g., germination medium)for the spores, or the liquid 122 can contact nutrient medium in a dryform (e.g., in a powdered or tablet form) to form nutrient medium, suchthat a mixture including the sterilized spores and nutrient medium isformed. The mixture can then be incubated prior to or during a detectionor assaying process, and the biological sterilization indicator 100 canbe interrogated for signs of spore growth.

Activation can further include moving the liquid 122 from the firstchamber 109 to the second chamber 111 via the first fluid path 160 whenthe container 120 is in the second state, and moving displaced gas(e.g., trapped air) out of the second chamber 111 via the second fluidpath 162 in response to, or to facilitate, moving the liquid 122 fromthe first chamber 109 to the second chamber 111 via the first fluid path160.

To detect a detectable change in the spores 115, the biologicalsterilization indicator 100 can be assayed immediately after the liquid122 and the spores 115 have been combined to achieve a baseline reading.After that, any detectable change from the baseline reading can bedetected. The biological sterilization indicator 100 can be monitoredand measured continuously or intermittently. In some embodiments, aportion of, or the entire, incubating step may be carried out prior tomeasuring the detectable change. In some embodiments, incubation can becarried out at one temperature (e.g., at 37° C., at 50-60° C., etc.),and measuring of the detectable change can be carried out at a differenttemperature (e.g., at room temperature, 25° C., or at 37° C.).

The readout time of the biological sterilization indicator 100 (i.e.,the time to determine the effectiveness of the sterilization process)can be, in some embodiments, less than 8 hours, in some embodiments,less than 1 hour, in some embodiments, less than 30 minutes, in someembodiments, less than 15 minutes, in some embodiments, less than 5minutes, and in some embodiments, less than 1 minute.

EMBODIMENTS

Embodiment 1 is a method of detecting a biological activity, comprising:

-   -   providing        -   a sample that may comprise a source of one or more            predetermined biological activities;        -   a first indicator system comprising a first indicator            reagent with a first absorbance spectrum, wherein the first            indicator reagent can be converted by a first predetermined            biological activity to a first biological derivative;        -   a second indicator system comprising a second indicator            reagent that is converted by a predetermined biological            activity to a second biological derivative with a second            emission spectrum; and        -   a substrate that receives and concentrates the first            indicator reagent from an aqueous mixture;    -   forming a first aqueous mixture comprising the sample, the first        indicator reagent, and the second indicator reagent;    -   bringing the first aqueous mixture into fluid communication with        the substrate to form a second aqueous mixture in which a        concentration of the first indicator reagent is lower than the        concentration of the first indicator reagent in the first        aqueous mixture; and    -   detecting a presence or absence of fluorescence from the second        biological derivative;    -   wherein the first absorbance spectrum includes detectable        absorbance in at least a portion of wavelengths present in the        second emission spectrum.

Embodiment 2 is the method of embodiment 1, wherein, detecting thepresence or absence of fluorescence from the second biologicalderivative comprises detecting the presence or absence of fluorescencein the second aqueous mixture.

Embodiment 3 is the method of embodiment for embodiment 2, furthercomprising observing the substrate to detect the first indicator reagentor the first biological derivative.

Embodiment 4 is the method of any one of the preceding embodiments,wherein a concentration of first indicator reagent in the first aqueousmixture is sufficient to prevent detection of an otherwise detectableamount of the second biological derivative.

Embodiment 5 is the method of any one of the preceding embodiments,further comprising providing a nutrient to facilitate growth of abiological cell, wherein forming the first aqueous mixture comprisesforming a mixture that includes the nutrient.

Embodiment 6 is the method of any one of the preceding embodiments,further comprising exposing the biological activity to a sterilant.

Embodiment 7 is the method of embodiment 6, wherein the sterilant isselected from a group consisting of steam, ethylene oxide, hydrogenperoxide, formaldehyde, and ozone.

Embodiment 8 is the method of any one of the preceding embodiments,further comprising exposing the biological activity to a temperatureshift for a period of time.

Embodiment 9 is the method of any one of the preceding embodiments,wherein the first indicator reagent comprises a chromophore, whereindetecting the first biological derivative comprises detecting a color

Embodiment 10 is the method of embodiment 9, wherein the first indicatorreagent comprises a chromogenic indicator.

Embodiment 11 is the method of embodiment 9 or embodiment 10, whereinthe first indicator reagent comprises a pH indicator or an enzymesubstrate.

Embodiment 12 is the method of embodiment 11, wherein the firstindicator reagent is selected from a group consisting of BromocresolPurple, Bromocresol Green, Congo Red, and Methyl Orange.

Embodiment 13 is the method of any one of the preceding embodiments,wherein the second indicator reagent comprises a fluorogenic compound.

Embodiment 14 is the method of embodiment 13, wherein the fluorogeniccompound comprises a fluorogenic enzyme substrate.

Embodiment 15 is the method of any one of the preceding embodiments,wherein detecting the presence or absence of the second biologicalderivative further comprises measuring a quantity of the secondbiological derivative.

Embodiment 16 is the method of any one of the preceding embodiments,wherein detecting the presence or absence of the first biologicalderivative further comprises measuring a quantity of the firstbiological derivative.

Embodiment 17 is the method of embodiment 16, wherein measuring thequantity of the first biological derivative comprises comparing anamount of color measured in a portion of the second aqueous mixture notassociated with the substrate to a color standard.

Embodiment 18 is the method of any one of the preceding embodiments,further comprising:

providing an instrument that detects the first indicator reagent or thesecond biological derivative; and

using the instrument to detect the first indicator reagent or the secondbiological derivative.

Embodiment 19 is the method of any one of the preceding embodiments,further comprising:

providing an instrument that detects the first indicator reagent and thesecond biological derivative; and

using the instrument to detect the first indicator reagent and thesecond biological derivative.

Embodiment 20 is a method of detecting a biological activity,comprising:

-   -   providing a biological sterilization indicator comprising;        -   a housing comprising first and second chambers;        -   a container containing a first aqueous liquid, the container            disposed in a first chamber, wherein at least a portion of            the container is frangible, the liquid comprising a first            indicator system comprising a first indicator reagent with a            first absorbance spectrum and a second indicator system            comprising a second indicator reagent that is converted by a            second predetermined biological activity to a second            biological derivative with a second emission spectrum,            wherein the first indicator reagent can be converted by a            first predetermined biological activity to a first            biological derivative, wherein the first absorbance spectrum            includes detectable absorbance in at least a portion of            wavelengths of the second emission spectrum;        -   a source of the second predetermined biological activity            disposed in a second chamber; and        -   a substrate that receives and concentrates the first            indicator reagent from the first aqueous liquid, the            substrate disposed in the housing;    -   bringing the first aqueous liquid into fluid communication with        the substrate to form a second aqueous liquid in which the        concentration of the first indicator reagent is lower than the        concentration of the first indicator reagent in the first        aqueous liquid; and    -   detecting a presence or absence of fluorescence from the second        biological derivative in the second aqueous mixture.

Embodiment 21 is the method of embodiment 20, wherein bringing the firstaqueous liquid into fluid communication with the substrate comprisesfracturing at least a portion of the frangible container.

Embodiment 22 is the method of embodiment 21, wherein the biologicalsterilization indicator further comprises a breaker disposed in thehousing and wherein fracturing the frangible container comprises urgingthe container and the breaker against one another.

Embodiment 23 is the method of any one of embodiments 20 through 21,wherein the housing of the biological sterilization indicator includes:

a first portion, and

a second portion adapted to be coupled to the first portion, the secondportion being movable with respect to the first portion, when coupled tothe first portion, between a first position and a second position;

wherein the method further comprises moving the second portion of thehousing from the first position to the second position.

Embodiment 24 is the method of embodiment 23, wherein the housingincludes a longitudinal direction, and wherein moving the second portionof the housing includes moving the second portion of the housing in thelongitudinal direction.

Embodiment 25 is the method of embodiment 23, further comprising movingthe container in the housing in response to moving the second portion ofthe housing from the first position to the second position.

Embodiment 26 is the method of embodiment 25, wherein moving thecontainer in the housing causes the container to fracture.

Embodiment 27 is a system to detect a predetermined biological activity,comprising:

-   -   a first indicator system comprising a first indicator reagent        with a first absorbance spectrum, wherein the first indicator        reagent can be converted by a first predetermined biological        activity to a first biological derivative;    -   a second indicator system comprising a second indicator reagent        that is converted by a predetermined biological activity to a        second biological derivative with a second emission spectrum;    -   a vessel configured to hold a liquid medium;    -   a substrate that receives and concentrates the first indicator        reagent from an aqueous mixture; and    -   an instrument configured to receive the vessel and to detect the        first indicator reagent or the second biological derivative;    -   wherein the first absorbance spectrum includes detectable        absorbance in at least a portion of wavelengths present in the        second emission spectrum.

Embodiment 28 is the system of embodiment 27, further comprising aprocessor.

Embodiment 29 is the system of embodiment 27 or embodiment 28, whereinthe instrument is further configured to regulate the temperature of theliquid medium.

Embodiment 30 is the system of any one of embodiments 27 through 29,wherein the instrument is configured to detect both the first indicatorreagent and the second biological derivative.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Reference Example 1 Absorbance Spectrum of Bromocresol Purple(BCP)

This reference example shows the absorbance spectrum of Bromocresolpurple.

Bromocresol purple obtained from Sigma Chemical Co., St. Louis, Mo.,(catalog number B-5880), was dissolved in phosphate buffered saline, pH7.3, at a concentration of 0.004. The solution was placed into a quartzcuvette and the u.v.-visible absorbance spectrum was scanned using the 1cm cuvette adapter provided with the TECAN INFINITE M200 Plate Reader(Tecan US, Durham, N.C.). The scan parameters are presented in Table 1.

The results are shown in the graph illustrated in FIG. 2. Absorbancepeaks can be seen at wavelengths of about 300 nm, about 380 nm, andabout 600 nm.

TABLE 1 Scan parameters for BCP absorbance spectrum. Mode FluorescenceTop Reading Emission Wavelength Start 380 nm Emission Wavelength End 650nm Emission Wavelength Step Size 2 nm Emission Scan Number 136Excitation Wavelength 350 nm Bandwidth (Em)  280 . . . 850: 20 nmBandwidth (Ex) (Range 1) 230 . . . 295: 5 nm Bandwidth (Ex) (Range 2)296 . . . 850: 9 nm Gain 80 Manual Number of Reads  10 Integration Time20 ms Lag Time 0 ms Settle Time 0 ms

Reference Example 2 Emission spectrum of 7-hydroxy-4-methylcoumarin(4-methylumbelliferone)

This reference example shows the emission spectrum of4-methylumbelliferone.

0.4-methylumbelliferone (4MU), catalog number m1381, obtained from SigmaChemical Co., St. Louis, Mo., was dissolved in phosphate bufferedsaline, pH 7.3, at a concentration of 0.004 mg/mL. The solution wasplaced into a quartz cuvette and the emission spectrum was recordedusing the 1 cm cuvette adapter provided with the TECAN INFINITE M200Plate Reader. The scan parameters are presented in Table 2.

The results are shown in the graph illustrated in FIG. 2. An emissionpeak can be seen at a wavelength of about 450 nm.

TABLE 2 Scan parameters for 4-methylumbelliferone emission spectrum.Mode Absorbance Wavelength Start 250 nm Wavelength End 800 nm WavelengthStep Size 2 nm Scan Number 276 Bandwidth (Range 1) 230 . . . 295: 5 nmBandwidth (Range 2) 296 . . . 1000: 9 nm  Gain 80 Manual Number of Reads 10 Settle Time 0 ms Part of Plate B1-B1

Reference Example 3 Effect of BCP on the Detection of 4MU

This reference example shows the effect of BCP on the detection of 4MUfluorescence when the two compounds are present in the same solution.

A stock solution of 4-methylumbelliferone (4MU), prepared as describedin Example 2, was serially diluted in the phosphate buffered saline tothe concentrations shown in Table 3. A stock solution of bromocresolpurple (BCP) was prepared in phosphate buffered saline, as described inExample 1. The bromocresol purple (0.03 mg/mL final concentration) wasmixed with the respective solutions of 4MU shown in Table 3. Triplicatealiquots (100 microliters/well) of each respective solution were loadedinto a 96-well plate and the fluorescence in each well was measuredusing a TECAN INFINITE M200 Plate Reader. The excitation wavelength was350 nm and the detection was at 420 nm. The results, listed as relativefluorescence units (RFU), are shown in Table 3. The data show that, atevery concentration of 4MU tested, the presence of bromocresol purple inthe solution resulted in a decrease in measurable fluorescence.

TABLE 3 Fluorescent detection of 4MU in the presence or absence of BCP4MU Concentration 4MU Without BCP 4MU With BCP (mg/mL) RFU RFU 0.0049725 5761 0.0004 927 582 0.00004 128 94 0.000004 50 40 0 41 37 Theresults are an average of three replicates. All values are reported inRelative Fluorescence units (RFUs).

Reference Example 4 Adsorption of BCP from a Liquid Medium

This reference example shows the adsorption of BCP from a growth mediumonto a substrate material.

A spore growth media solution was prepared consisting of 17 g of abacteriological peptone, 0.17 g of L-alanine and 0.03 g bromocresolpurple pH indicator dye, per liter of water. The pH of the nutrientmedium solution was adjusted to 7.6 with 0.1 N sodium hydroxide.

To each of 60 borosilicate glass tubes (12 mL, VWR Cat #53283-802) wasadded 1.0 mL of the prepared growth media and capped with linerless capclosures (VWR Cat #66010-680).

Two different substrate materials were evaluated: GE charged nylon(MAGNAPROBE 0.45 micron charged nylon membrane, part number NP0HY00010,available from GE Osmonics Labstore, Minnetonka, Minn.) and paper(Whatman Grade 1 Chr cellulose chromatography paper, available fromWhatman Inc. USA, Piscataway, N.J.).

Twenty strips of each of the two substrate materials were cut to size, 4mm×10 mm. All the strips were pre-sterilized by placing them in aPropper CHEX-ALL II Instant Sealing Pouch (Propper, Manufacturing Inc.,Long Island City, N.Y.) and sterilizing them for 30 minutes in a steamliquid cycle at 121° C. in an AMSCO sterilizer (Steris, Mentor, Ohio).

The sterilized substrate strips were aseptically removed from the pouchand transferred to the glass tubes, five strips of the nylon substrateper each of 20 tubes and five strips of the paper substrate per each of20 different tubes.

Spore strips were acquired from disassembled 1292 ATTEST Rapid ReadoutBiological Indicators Steam Sterilizers (3M, St. Paul, Minn.),containing G. stearothermophilus spores, (ATCC 7953). The spore stripswere cut into equal quarters, each approximately 6.4 mm×6.4 mm, andadded to glass tubes according to Table 4 and further described below.One (6.4 mm×6.4 mm) piece of a 1292 ATTEST spore strip was added to eachof 10 glass tubes, each containing 5 pieces of the nylon substrate andgrowth media. One piece of the spore strip was added to each of 10 glasstubes, each tube containing 5 pieces of the Whatman paper and growthmedia. One piece of spore strip was added to each of 10 glass tubes,each tube containing only growth media, no substrate. No spore strippiece was added to the remaining 30 tubes: 10 tubes containing 5 piecesof nylon substrate, 10 tubes containing 5 pieces of the paper substrate,and 10 tubes containing no substrate.

TABLE 4 Preparation of Samples for Example 4 Number Growth 5 strips ofSpore Sample of tubes Media Substrate Strip 1. Nylon + spores 10 yesnylon Yes 2. Nylon w/o spores 10 yes nylon None 3. Paper + spores 10 yespaper Yes 4. Paper w/o Spores 10 yes paper None 5. Control - Nosubstrate + 10 yes none Yes spores 6. Control - No substrate w/o 10 yesnone None spores

Two tubes of each of the above samples were selected for the followingobservations and analyses at 1 minute time point. The color of the nylonor paper substrate material while in the tube was compared to the colorof the surrounding liquid growth media as to whether the substrate wasdarker or lighter than the media. The color of substrate materials wereobserved and recorded when taken out of the glass tubes containinggrowth media.

The nylon and paper substrate strips were removed from the tubes andplaced on a KIMWIPE (Kimberly-Clark) before densitometry readings weretaken using an X-Rite 530P densitometer (X-Rite, Grand Rapids Mich.).The optical density setting on the X-Rite 530P densitometer was set to“color” to provide the V filter results. The X-Rite densitometer was setto “compare” for substrate results of ΔE with Pantone 2665U and 102Uselected. The CIE76 formula was used to calculate the ΔE at eachPantone. The ΔE value is the distance in L*A*B colorspace from ameasured point to a reference value, a Pantone color. A lower ΔEindicates a measured color is closer to the reference value. A value ofabout 2.5 ΔE's is about the minimum threshold for a human eye todifferentiate color. The two reference values used were Pantone 2665U (alight purple) and Pantone 102U (bright yellow). Note that, because thesetwo values are not diametrically opposed on the “color wheel”, anincrease in ΔE at 2665 U does not necessarily mean an exact decrease inΔE at 102 U. In other words ΔE at 2665 U only indicates whether or notsomething got more “purple”, not whether or not something got more“yellow”.

The color of the media in each tube was also observed and recorded(Table 5). In triplicate, an amount of 200 μL of media was removed fromeach tube and placed in a 96 well plate (COSTAR CLS-3603-48EA blacktissue culture treated 96 well plate with clear bottom) and the opticaldensity at 590 nm and 430 nm was measured with a SYNERGY 4spectrophotometer with Gen 5 software. OD measurements were taken usingMonochromator, (BioTek, Winooski, Vt.).

The remaining tubes were incubated at 56° C. At each of the followingtimes: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubesof each sample were removed from the incubator, visually observed andinstrumentally measured as described above.

TABLE 5 Color Observations of Media Sample 1 min 30 min 1 hr 4 hrs 24hrs 1. Nylon + spores Purple Purple Purple Purple Yellow 3. Paper +spores Purple Purple Purple Purple Yellow 5. Control - No Purple PurplePurple Purple Yellow substrate + spores 2. Nylon w/o spores PurplePurple Purple Purple Purple 4. Paper w/o Spores Purple Purple PurplePurple Purple 6. Control - No Purple Purple Purple Purple Purplesubstrate w/o spores

The media in all vials remained purple until after the 4 hour reading.Those samples without spores remained purple after 24 hours. All sampleswith spores had turned a visually yellow color by 24 hours due to growthof cells leading to a decrease in pH of the media, indicated by the BCPpH indicator dye.

At each time interval, before the substrate was removed from the media,the color of the substrate was compared to that of the media (see Table6). If there was any difference between the two colors the differencewas documented. In all instances when the nylon substrate was used asthe substrate, the substrate appeared a darker shade of the color thanthe surrounding media. In all instances when paper was used as thesubstrate, the substrate appeared as a lighter shade of the color of thesurrounding media. These results show that the nylon substrate issuperior to the paper substrate in receiving and concentrating theindicator reagent.

TABLE 6 Substrate Color vs. Media Color 1 0.5 1 4 24 Sample minute hourshour hours hours 1. Nylon + Spores Darker Darker Darker Darker DarkerYellow 2. Nylon w/o spores Darker Darker Darker Darker Darker 3. Paper +Spores Lighter Lighter Lighter Lighter Lighter Yellow 4. Paper w/oSpores Lighter Lighter Lighter Lighter Lighter

In most instances “Darker” meant that the substrate was a visibly darkerpurple color than the media, with the exception of 24 hrs nylon withspores, which was a darker yellow color. In most instances “Lighter”meant that the substrate was a visibly lighter purple color than themedia, with the exception of 24 hrs paper with spores, which was alighter yellow color.

For the samples with spores, the OD measurement at 590 nm at 24 hourswill not show the differences in the intensity of the yellow color.Therefore, only the OD values taken at 430 nm at 24 hours wereevaluated.

TABLE 7 Average Optical Density of Media at 430 nm at 24 hours Sample 24hrs 1. Nylon + spores 0.271 3. Paper + spores 0.827 5. Control - nosubstrate + spores 0.835

The 24 hour readings at 430 nm of the media samples with spores in thepresence of paper substrate and the media sample with no substrate(Control) with spores, both have similar values of OD of 0.827 and 0.835respectively, as shown in Table 7. However, the media sample with sporesin the presence of nylon had an OD of only 0.271; which is 0.5 OD unitsless than the control or the sample with the paper substrate. This showsthat the intensity of the yellow color of the media in the presence ofnylon was reduced due to the nylon substrate receiving and concentratingthe indicator reagent.

TABLE 8 Average Optical Density of Media at 590 nm Sample 1 min 30 min 1hr 4 hr 24 hrs 1. Nylon with spores 1.124 1.122 0.698 1.063 *** 3. Paperwith spores 1.404 1.786 1.440 1.697 *** 5. Control - No substrate with1.402 1.801 1.463 1.776 *** spores 2. Nylon w/o spores 1.158 1.136 0.6531.102 1.122 4. Paper w/o Spores 1.435 1.716 1.468 1.863  1.708* 6.Control - No substrate w/o 1.345 1.797 1.465 1.828 1.812 spores Allvalues represent n = 6 (3 readings × 2 tubes). *n = 5 readings: 3readings for tube 1 and 2 reading for tube 2. ***Samples are yellow incolor and therefore OD at 590 nm does not accurately measure the colorof the media.

The absorbance of the control with no substrate (with and withoutspores) at 1 minute was considered the initial baseline OD measurementfor the media. Table 8 shows that even at 1 minute the OD at 590 nm ofthe sample media, with spores, in the presence of the nylon, (1.124) wasless than the OD of the sample media in the presence of the paper withspores (1.404) or the Control with spores (1.402). This differenceindicates that the intensity of the purple color of the media wasalready reduced due to the nylon substrate rapidly receiving andconcentrating the BCP indicator reagent. At 24 hours the OD at 590 nm ofthe sample media without spores in the presence of the nylon was 1.122,which is much lower than the OD of the media in the presence of paper(1.708) or the OD of the Control sample without spores, 1.812.

TABLE 9 Pantone Color of Substrate at 24 hours 1. Nylon substrate +spores Yellow Pantone 102U 2. Nylon substrate w/o spores Purple Pantone1345U 3. Paper substrate + spores Yellow Pantone 100U 4. Paper substratew/o spores Purple Pantone 256U Initial Purple Media Color Purple Pantone2665U Yellow Media Color Yellow Pantone 102U

TABLE 10 Average Densitometry Reading of Substrate using V filter Sample0 min 1 min 0.5 hrs 1 hrs 4 hrs 24 hrs 1. Nylon + spores 0.05 0.17 0.300.22 0.26 *** 2. Nylon w/o spores 0.05 0.20 0.24 0.28 0.26 0.26 3.Paper + spores 0.11 0.12 0.26 0.16 0.12 *** 4. Paper w/o spores 0.110.11 0.15 0.14 0.11 0.18 *** Substrate samples with spores at 24 hoursare yellow in color and V filter does not accurately measure the colorof the substrate.

TABLE 11 Average Densitometry Reading of Substrate: Δ E from Pantone2665U (Purple) Sample 0 min 1 min 0.5 hrs 1 hrs 4 hrs 24 hrs 1. Nylon +spores 68.18 64.48 63.57 64.36 56.42 *** 2. Nylon w/o spores 68.18 66.3758.47 74.45 54.62 57.49 3. Paper + spores 66.23 68.18 61.27 67.06 66.96*** 4. Paper w/o spores 66.23 68.70 65.29 63.69 67.45 59.31 ***Substrate samples with spores at 24 hours are yellow in color and Vfilter does not accurately measure the color of the substrate.

TABLE 12 Average Densitometry Reading of Substrate: ΔE from Pantone 102U(Yellow) Sample 0 min 24 hrs Nylon substrate + Spores 83.78 56.83 Papersubstrate + Spores 83.67 76.15

The above tables show the densitometry readings of the substrates afterexposure to media (with and without spores) for varying lengths of time.The time 0 reading for each substrate is the initial densitometryreading before the substrate sample is placed into the media. Whenevaluating the substrates that are purple, the V filter and the Δ E(Pantone 2665U) showed the most contrast. The average densitometryreadings with the V filter for the nylon substrate as shown in Table 10increased and remained elevated throughout the experiment (with the onlyexception being when the substrate was yellow at the 24 hour time pointfor the “with spore” sample). In contrast, the densitometry readings forthe paper substrate remained fairly constant across the time points.Likewise, the nylon substrate Δ E (Pantone 2665U) value shown in Table11 generally decreased throughout the experiment (with the onlyexception being when the substrate was yellow at the 24 hour time pointfor the “with spore” sample). This indicated the nylon substrate wasreceiving and concentrating the BCP indicator reagent. While incontrast, the Δ E (Pantone 2665U) value for the paper substrate remainedfairly constant.

Table 12 illustrates that at the 24 hour time point the ΔE (Pantone102U) value for the nylon substrate was considerably lower than the Δ E(Pantone 102U) value for the paper substrate, indicating that the nylonsubstrate was closer to the pantone 102U color (more bright yellow) thanthe paper substrate.

Reference Example 5 Nylon Substrate Adsorption of BCP from a LiquidMedium after Two 24 hr Incubations

This reference example shows the adsorption of BCP from a liquid growthmedium onto a nylon substrate.

The same media and components used in Example 4 were used in Example 5.To each of 4 glass tubes was added 1.0 mL of the prepared growth media.One piece of a 1292 ATTEST spore strip cut to approximately 6.4 mm×6.4mm was added to each glass tube. The tubes were placed in an incubatorat 56° C. for 24 hours to promote the growth of the G.stearothermophilus cells. After the 24 hours incubation, five (5) strips(each cut to 4 mm×10 mm) of the nylon substrate were added to two (2) ofthe tubes. The tubes were placed in an incubator at 56° C. for another24 hours. After the second 24 hour incubation period (24 hours after theaddition of the nylon substrate) to the tubes, the following analyseswere performed. The nylon substrate pieces were removed from the tubes,placed on a KIMWIPE and densitometry readings of the substrate stripswere taken. From each tube three aliquots of 200 μL were taken andplaced into in a 96 well plate. The optical density at 430 nm of themedia was measured.

TABLE 13 Average OD of Media at 430 nm at 48 hours; 24 hours after nylonsubstrate Sample Average OD Media containing nylon substrate + spores0.365 Control media - no substrate + spores 1.241 n = 12 (3 readingsfrom each of 4 tubes) n = 3 (1 reading from the one control tube)

Table 13 shows the decrease in the OD at 430 nm of the media 24 hoursafter the nylon substrate was added to the tubes, compared to thecontrol, where no substrate was added. The difference in the ODmeasurement between the two samples indicates the difference in theamount of yellow present in the sample media. This shows that theintensity of the yellow color of the media in the presence of nylon wasreduced due to the nylon substrate receiving and concentrating theindicator reagent.

TABLE 14 Average Densitometry Reading of Substrate: ΔE from Pantone 102U(Yellow) Sample Avg. ΔE (102U) Nylon substrate + spores after 24 hrs inmedia 37.86 Nylon substrate before media 83.78 n = 10 (5 strips of nylonsubstrate × 2 tubes)

Table 14 shows the ΔE (102U) value of the nylon substrate 24 hours afterbeing added to a tube of media with spores that had already beenincubated for 24 hours. This was compared to nylon substrate that wasnot placed into media. The difference in the ΔE measurements between thetwo samples indicates that the substrate exposed to (yellow) media withgrowth is closer in color to pantone 102U (bright yellow) than thesubstrate not exposed to the media.

Reference Example 6 Absorption of BCP from a Liquid Medium by VariousSubstrates

This reference example shows the adsorption of BCP from a liquid growthmedium onto various substrate materials.

A spore growth media solution was prepared consisting of 17 grams of abacteriological peptone C, 0.17 grams of L-alanine and 0.03 gramsbromocresol purple (BCP) pH indicator dye, per liter of water. The pH ofthe nutrient medium solution was adjusted to 7.6 with 0.1 N sodiumhydroxide.

To each borosilicate glass tube (12 mL, VWR Cat #53283-802) was added1.0 mL of the prepared growth media and capped with linerless capclosures (VWR Cat #66010-680).

Four different substrate materials were evaluated: (1) GE charged nylon(MAGNAPROBE 0.45 micron charged nylon membrane, part number NP0HY00010,available from GE Osmonics Labstore, Minnetonka, Minn.); (2) BIO-RADhigh-strength nylon membrane positively charged with quaternary aminegroups (ZETA-PROBE GT Genomics, Cat#162-0196, available from BIO-RADLifeSciences, Hercules, Calif.); (3) 0.2 μM nitrocellulose (Cat#LC-2000, available from Invitrogen Corporation Carlsbad, Calif.), and(4) 0.2 μM polyvinylidene difluoride (PVDF) membrane (Cat# LC-2002,available from Invitrogen Corporation Carlsbad, Calif.). Several stripsof each of the substrate materials were cut to size: 4 mm×10 mm, enoughfor one (1) strip for each glass tube.

All the strips were pre-sterilized by placing them in a Propper CHEX-ALLII Instant Sealing Pouch (Propper, Manufacturing Inc., Long Island City,N.Y.) and sterilizing them for 30 minutes in a steam liquid cycle (at121° C.) in an AMSCO sterilizer (Steris, Mentor, Ohio). The strips werethen aseptically transferred to each tube. Two tubes of each substratewere evaluated along with two control tubes that contained no substrate.

The following observations and analyses were performed at 0 time, 30minutes, 1 hour, 4 hours and 24 hour time points: (1) the color of thesubstrate material in each tube was compared to the color of thesurrounding media of the same tube. (darker or lighter), (2) thesubstrate material was removed from the tube, placed on a KIMWIPE toblot dry and then densitometry readings were taken with the V filter asdescribed above, (3) removed 200 μL of the media from each tube andtransferred in triplicate into a 96 well plate (COSTAR CLS-3603-48EAblack tissue culture treated 96 well plate with clear bottom) and theoptical density of the media at 590 nm. was measured with a SYNERGY 4spectrophotometer with Gen 5 software. OD measurements were taken usinga Monochromator, (BioTek, Winooski, Vt.).

The remaining tubes were incubated at 56° C. At each of the followingtimes: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubesof each sample were removed from the incubator, visually observed andinstrumentally measured as describe above.

TABLE 15 Substrate Color vs. Media Color for Various Substrate s Bio-RadGE ZETA- MAGNAPROBE PROBE Invitrogen Invitrogen Time Point Nylon NylonNitrocellulose PVDF 0 hr Lighter Lighter Lighter Lighter 0.5 hr   DarkerDarker Lighter Lighter 1 hr Darker Darker Lighter Lighter 4 hr DarkerDarker Lighter Lighter 24 hr  Darker Darker Lighter Lighter Darker =Substrate was visibly darker purple color than the media Lighter =Substrate was visibly lighter purple color than the media

At each reading before the substrate was removed from the media, thecolor of the media was visually compared to that of the substrate. Thedifference between the color of the substrate and the color of the mediawas observed and reported in Table 15. After 30 minutes in contact withthe media, both nylon substrate materials were visibly darker than themedia and remained darker throughout the entire experiment.

TABLE 16 Average Densitometry Reading of Various Substrates after Mediawith BCP Bio-Rad GE ZETA- MAGNAPROBE PROBE Invitrogen Invitrogen TimePoint″ Nylon Nylon Nitrocellulose PVDF 0 hr 0.300 0.425 0.050 0.035 0.5hr   0.965 0.735 0.195 0.030 1 hr 0.930 0.785 0.255 0.025 4 hr 1.0350.720 0.220 0.035 24 hr  1.015 0.735 0.240 0.040

Table 16 shows the Densitometry readings of the substrate materialsafter exposure to media for varying lengths of time. The time 0 readingfor each substrate is the initial densitometry reading within 30 secondsof the substrate being placed into the media. In all instances the nylonsubstrates densitometry increased within 30 minutes and remainedelevated throughout the experiment.

TABLE 17 O.D. at 590 nm of Media in Presence of Various SubstrateMaterials GE Bio-Rad MAGNA- ZETA- Control Time PROBE PROBE InvitrogenInvitrogen (Media Point Nylon Nylon Nitrocellulose PVDF only) 0 hr 1.9721.952 2.012 1.988 1.957 0.5 hr   1.535 1.762 1.981 1.985 1.965 1 hr1.166 1.662 2.143 1.970 1.990 4 hr 1.108 1.704 2.071 1.995 1.958 24 hr 0.935 1.842 2.217 2.329 2.156

Table 17 shows the average optical density reading of the media removedfrom the tube containing each substrate material at the specified time.It is noticeable that at each time point, the OD for the media which wasin the presence of either nylon substrate was lower than the OD readingfor the media containing either the nitrocellulose or the PVDF.Additionally, the nitrocellulose or the PVDF show very little change inOD reading and are quite similar to the Control OD values.

Reference Example 7 Substrate Absorption of Methyl Red (MR) from aLiquid Medium

This reference example shows the adsorption of BCP from a liquid growthmedium onto various substrate materials.

A spore growth media solution was prepared consisting of 17 grams of abacteriological peptone, 0.17 grams of L-alanine and 0.03 grams methylred pH indicator dye, per liter of water. The pH of the nutrient mediumsolution was adjusted to 4.2 with 0.1 N hydrochloric acid.

To each borosilicate glass tubes (12 mL, VWR Cat #53283-802) was added1.0 mL of the prepared growth media and capped with linerless capclosures (VWR Cat #66010-680).

Two different substrate materials were evaluated: GE charged nylon(MAGNAPROBE 0.45 micron charged nylon membrane, part number NP0HY00010,available from GE Osmonics Labstore, Minnetonka, Minn.), and BIO-RADhigh-strength nylon membrane positively charged with quaternary aminegroups (Zeta-Probe GT Genomics, Cat#162-0196, available from Bio-RadLifeSciences, Hercules, Calif.). Several strips of each substratematerial were cut to size: 4 mm×10 mm, enough for one (1) strip for eachglass tube.

All the strips were pre-sterilized by placing them in a Propper CHEX-ALLII Instant Sealing Pouch (Propper, Manufacturing Inc., Long Island City,N.Y.) and sterilizing them for 30 minutes in a steam liquid cycle (at121° C.) in an AMSCO sterilizer (Steris, Mentor, Ohio). The strips werethen aseptically transferred to each tube.

The following observations and analyses were performed for two tubes ofeach substrate at 0 time, 30 minutes, 1 hour, 4 hours and 24 hour timepoints: (1) the substrate material was removed from the tube, placed ona KIMWIPE to blot dry and then densitometry readings were taken with theV filter as performed above, (2) the color of the substrate material ineach tube was compared to the color of the surrounding media of the sametube. (darker or lighter).

The remaining tubes were incubated at 56° C. At each of the followingtimes: 30 minutes, 1 hour, 4 hours and 24 hours of incubation; 2 tubesof each sample were removed from the incubator, visually observed andinstrumentally measured as describe above.

TABLE 18 Average Densitometry Reading of Substrate after Methyl Red, Vfilter GE Bio-Rad MAGNAPROBE ZETA-PROBE Time Point Nylon Nylon 0 hr0.160 0.200 0.5 hr   0.285 0.330 1 hr 0.325 0.376 4 hr 0.205 0.450 24hr  0.470

The above tables show the densitometry readings of the substrates afterexposure to media for a varying length of time. The time 0 reading foreach substrate is the initial densitometry reading within 30 seconds ofthe substrate being placed into the media. In all instances the Nylonsubstrates densitometry increased within 30 minutes and remainedelevated throughout the experiment.

TABLE 19 Substrate Color vs. Media Color after Methyl Red GE Bio-RadMAGNAPROBE ZETA-PROBE Time Point Nylon Nylon 0 hr Lighter Lighter 0.5hr   Darker Darker 1 hr Darker Darker 4 hr Darker Darker 24 hr  DarkerDarker Darker = Substrate was visibly darker than the media Lighter =Substrate was visibly lighter than the media

At each reading before the substrate materials were removed from themedia, the color of the media was compared to that of the substrate (seeTable 19). The difference between the color of the substrate and thecolor of the media was observed and reported. After 30 minutes incontact with the media, both of the nylon substrate materials werevisibly darker than the media and remained darker throughout the entireexperiment.

Reference Example 8 Inhibition of Acridine Orange (AO) Detection withBCP and Methyl Red

This reference example shows the effect of BCP and methyl red on thedetection of acridine orange fluorescence when one of the pH indicators(i.e., BCP or MR) is present in a solution with acridine orange.

A spore growth media solution was prepared consisting of 17 grams of abacteriological peptone and 0.17 grams of L-alanine. A volume of 200 μLof the growth media was added to each well in two (2) 96 well plates.

A dilution series of pH indicator solutions was made for both Methyl Red(MR) and Bromocresol Purple (BCP) starting at 4.8 g/L and diluted downto 0.75 g/L. A dilution series of acridine orange was made starting at1:50 and diluting down to 1:800.

In plate #1, 20 μL the appropriate dilution of BCP was added to each rowof the plate and 20 μL of the appropriate dilution of acridine orange(AO) was added to each column of plate #1. In plate #2, 20 μL of theappropriate dilution of Methyl Red was added to each row of the plateand 20 μL of the appropriate dilution of acridine orange (AO) was addedto each column of plate #2. See Table 20 for set up of plate #1 andplate #2.

TABLE 20 Set up for 96 Well Plate #1 BCP and Plate #2 MR Column: InitialDilution of OA A B C D E F Row: Initial Conc. of BCP 1:50 1:100 1:2001:400 1:800 No or MR AO AO AO AO AO AO Row 1: 4.8 g/L BCP or MR — — — —— — Row 2: 2.4 g/L BCP or MR — — — — — — Row 3: 1.2 g/L BCP or MR — — —— — — Row 4: 0.6 g/L BCP or MR — — — — — — Row 5: 0.3 g/L BCP or MR — —— — — — Row 6: 0.15 g/L BCP or MR — — — — — — Row 7: 0.075 g/L BCP or MR— — — — — — Row 8: No pH indicator — — — — — —

Plates #1 and #2 were placed into the SYNERGY 4 spectrophotometer andabsorbance readings taken at 590 nm. Additionally, fluorescenceexcitation/emission readings at 435 nm/530 nm were also collected (seeTables 21A-B).

TABLE 21A Inhibition of Acridine Orange Detection with BCP 1:50 AO 1:100AO 1:200 AO Row 590 435/530 590 435/530 590 435/530 Initial [BCP] nm nmnm nm nm nm 4.8 g/L BCP ** 94.5 ** 111.0 ** 88.0 2.4 g/L BCP 3.709 173.0** 176.5 3.991 163.0 1.2 g/L BCP 2.189 267.0 2.243 299.5 2.427 338.0 0.6g/L BCP 1.244 399.0 1.236 505.5 1.305 531.5 0.3 g/L BCP 0.726 552.00.713 717.0 0.645 717.0 0.15 g/L BCP 0.401 635.0 0.408 821.0 0.400 904.50.075 g/L BCP 0.264 670.5 0.274 912.0 0.241 958.0 0 BCP 0.139 758.50.128 1059.0 0.101 1114.5 ** Signal above detection threshold ofinstrument

TABLE 21B Inhibition of Acridine Orange Detection with BCP Row 1:4001:800 No AO Initial [BCP] 590 435/530 590 435/530 590 435/530 4.8 g/LBCP ** 73.5 ** 45.5 ** 7.5 2.4 g/L BCP ** 105.5 ** 80.5 ** 16.0 1.2 g/LBCP 2.194 255.5 2.488 184.0 2.190 29.5 0.6 g/L BCP 1.325 445.0 1.318289.0 1.223 48.5 0.3 g/L BCP 0.684 607.0 0.653 418.0 0.696 67.5 0.15 g/LBCP 0.389 746.5 0.373 486.5 0.402 84.5 0.075 g/L BCP 0.269 838.0 0.234535.0 0.278 97.0 0 BCP 0.117 941.0 0.098 635.5 0.123 116.0 ** Signalabove detection threshold of instrument

For all acridine orange concentrations, as the amount of BromocresolPurple in solution decreased the signal generated by the acridine orangeincreased. In other words the presence of BCP masked the acridine orangesignal. For example, for the row with an initial BCP concentration of0.3 g/L, between about 27-36% of the acridine orange fluorescence signalis lost, compared to the row with 0 BCP.

TABLE 22A Inhibition of Acridine Orange Detection with Methyl Red 1:50AO 1:100 AO 1:200 AO Row 590 435/530 590 435/530 590 435/530 Initial[MR] nm nm nm nm nm nm 4.8 g/L MR ** 364.0 ** 473.5 ** 540.5 2.4 g/L MR2.211 484.5 3.835 434.5 1.848 470.0 1.2 g/L MR 2.466 462.0 1.102 672.50.889 579.0 0.6 g/L MR 0.733 656.0 1.238 768.5 1.075 612.0 0.3 g/L MR0.571 722.0 0.412 996.0 1.693 882.0 0.15 g/LMR 1.123 664.0 0.644 940.50.882 891.5 0.075 g/L MR 0.588 694.0 0.377 1036.0 0.367 1066.5 0 MR0.566 741.5 0.390 1048.5 0.303 1053.5 ** Signal above detectionthreshold of instrument

TABLE 22B Inhibition of Acridine Orange Detection with Methyl Red 1:400AO 1:800 AO No AO Row 590 435/530 590 435/530 590 435/530 Initial [MR]nm nm nm nm nm nm 4.8 g/L MR 0.517 390.0 ** 329.0 ** 27.0 2.4 g/L MR1.806 476.5 3.764 248.0 3.236 35.0 1.2 g/L MR 3.938 401.5 2.821 327.02.488 48.5 0.6 g/L MR 1.883 599.0 1.571 456.5 1.604 71.0 0.3 g/L MR0.892 733.0 1.398 478.5 0.879 81.5 0.15 g/L MR 0.751 741.5 0.441 543.00.330 93.5 0.075 g/L MR 0.292 891.5 0.293 622.0 0.245 109.0 0 MR 0.275876.0 0.232 658.0 0.247 116.5 ** Signal above detection threshold ofinstrument

Like BCP, the methyl red also masked the acridine orange fluorescencesignal. The higher the concentration of methyl red the lower thedetected fluorescence signal of acridine orange. For example, for therow with an initial methyl red concentration of 0.3 g/L, between about3-27% of the acridine orange fluorescence signal is lost, compared tothe row with no methyl red (see Tables 22A-B).

Examples 1-3 Detecting a Biological Activity

3M ATTEST 1291 Rapid Readout Biological Indicators are obtained from 3MCompany, St. Paul, Minn. Charged nylon membrane (MAGNAPROBE 0.45 microncharged nylon membrane, part number NP0HY00010) is obtained from GEOsmonics Labstore (Minnetonka, Minn.).

The caps of the biological indicators are removed and the glass ampulesare removed by inverting the biological indicator tube. The ampules areset aside for later use. The nylon membrane is cut into small strips(0.5 cm×2 cm). A strip is placed (lengthwise) adjacent the wall at thebottom of the biological indicator tubes and the glass ampule isreplaced in each tube. The caps are carefully replaced on each tube.

The modified biological indicators are subjected to exposure to steamfor varying lengths of time (shown in Table 23). The steam exposure isconducted at 270° F./132° C. Gravity Steam in a H&W Steam Resistometer(available from H&W Technology LLC, Rochester, N.Y.). Following exposureto the steam, the biological indicators are allowed to cool and theampules are crushed in the biological indicators according to themanufacturer's instructions.

The ampules are placed in an incubator at 56° C. and periodically (e.g.,after 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120minutes, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24hours, 36 hours, 48 hours, and/or 72 hours) removed to observe the colorof the liquid medium, the color of the nylon membrane, and thefluorescence of the liquid medium. The fluorescence can be detectedvisually by illuminating the tubes with a hand-held ultraviolet lightsource or, alternatively, the tubes (or the liquid therefrom) can beplaced in a suitable fluorometer to measure the fluorescence.

The biological indicators that are subjected to little or no steamexposure (e.g., 0-1 minutes steam exposure) will show conversion of thepH indicator (bromocresol purple) from purple to yellow in the broth andon the nylon membrane. These biological indicators will also showsubstantial conversion of the fluorogenic enzyme substrate to afluorescent end product (4-methylumbelliferone).

The biological indicators that are subjected to a lethal steam exposure(e.g., ≧15 minutes) will show accumulation of the bromocresol purple onthe nylon membrane, but will not show substantial conversion of theindicator from purple to yellow. These biological indicators will notshow substantial conversion of the fluorogenic enzyme substrate to afluorescent end product.

TABLE 23 Substrate in first position - Fluorescence Example SteamExposure No. (minutes) 1 0 2 1 3 15

Reparatory Example 1 Preparation of a Biological Sterilization Indicator(BI)

To exemplify the present disclosure, several biological sterilizationindicators (BIs) were prepared, according to the descriptions providedabove and as shown in FIGS. 4-7. The particular details of the BIs usedin the examples are provided below.

As shown in FIGS. 4-7, the biological sterilization indicator 100included a housing 102, which contained a first portion 104 (e.g., ahollow tube) and a second portion 106 (e.g., a cap) that were coupledtogether to provide a self-contained biological sterilization indicator.The cap was molded polypropylene with general dimensions ofapproximately 21 mm long by 14 mm in diameter. The first portion 104(hollow tube) was molded polycarbonate, with the general dimensions ofabout 52 mm long and 12 mm in diameter at top, with the shape shown inFIGS. 4-6. The total volume of the first portion 104 (e.g., a hollowtube) was approximately 3 mL.

As shown in FIGS. 4-6, the second portion (cap) 106 of the housing 102included 6 apertures or openings 107, which provided fluid communicationbetween the interior of the housing 102 (e.g., the reservoir 103) andambience. A filter paper material (not shown in FIGS. 4-6) which actedas a barrier; was positioned in the sterilant path over the apertures107 and held in place with a pressure sensitive adhesive backed paperlabel. The filter paper material was the same material present in thecap of currently available 3M ATTEST 1291 Rapid Readout BiologicalIndicators for Steam Sterilizers, available from 3M Company of St. Paul,Minn.

The biological sterilization indicator 100 further included a frangiblecontainer 120 that contained liquid growth media 122. The frangiblecontainer 120 was made of borosilicate glass and contained the sporegrowth media. The media consisted of a modified Tryptic Soy Broth (TSB)containing a pH indicator bromocresol purple, and a fluorescent enzymesubstrate 4-Methylumbelliferyl-alpha-D-glucoside. The ampoule wasapproximately 40 mm long by about 4 mm in diameter and heldapproximately 500 μL of media liquid. The liquid growth media 122 wasthe same media used in product currently available from 3M Company ofSt. Paul, Minn. as 3M ATTEST 1291 Rapid Readout Biological Indicatorsfor Steam Sterilizers.

As shown in FIGS. 4-7, the liquid media container 120 was held in placewithin the biological sterilization indicator 100 by an insert 130. Theinsert (also called a breaker) 130 served to both hold the container 120in place and function to facilitate the controlled breakage of thecontainer 120, which occurs during an activation step of the BI, whenthe second portion 106 is pushed down to break the liquid mediacontainer 120. The insert 130 was a molded polycarbonate structure withapproximate dimension of 22 mm long by 9 mm wide.

The second portion 106 had a seal projection 156 positioned to contactthe first end 101 of the first portion 104, at open upper end 157 of thefirst portion 104 to close or seal (e.g., hermetically seal) thebiological sterilization indicator 100 after activation.

The biological sterilization indicator 100 further included G.stearothermophilus spores (ATCC 7953) 115 positioned in fluidcommunication with the first portion 104. The spores 115 were depositedin a spore reservoir 136 of a polypropylene spore carrier 135 (9 mm×4mm) The spores 115 were deposited directly onto the polypropylenesurface, and the spore reservoir 136 had a volume of approximately 15μt.

The housing 102 included a lower portion 114 (that at least partiallydefined a first chamber 109) and an upper portion 116 (that at leastpartially defined a second chamber 111), which were partially separatedby an inner partial wall or ledge 118, in which was formed an opening117 that provided fluid communication between the first chamber 109 andthe second chamber 111. The second chamber 111 was adapted to house thespores 115. The first chamber 109 was adapted to house the frangiblecontainer 120, particularly before activation. The wall 118 was angledor slanted, at a non-zero and non-right angle with respect to thelongitudinal direction of the housing 102, as shown in FIGS. 4-7.

The second chamber 111, which can also be referred to as the “sporegrowth chamber” or “detection chamber,” included a volume to beinterrogated for spore viability to determine the efficacy of asterilization process.

The liquid media container 120 was positioned and held in the firstchamber 109 during sterilization and when the container 120 wasunfractured. The spores 115 were housed in the second chamber 111 and influid communication with ambience during sterilization. The sterilantmoved into the second chamber 111 (e.g., via the first chamber 109)during sterilization. Afterwards, the liquid media 122 moved into thesecond chamber 111 (e.g., from the first chamber 109) during activation,when the container 120 was fractured and the liquid 122 was releasedinto the interior of the housing 102.

The first chamber 109 had a volume of about 2800 microliters (empty ofall internal components). The cross-sectional area of the first chamber109, immediately above the wall 118 was approximately 50 mm². The secondchamber 111 had a volume of about 210 microliters. The cross-sectionalarea of the second chamber 111, immediately below the wall 118, wasapproximately 20 mm².

The biological sterilization indicator 100 further included a substrate119. The substrate 119 was approximately 9 mm×8 mm in size, and wasdimensioned to rest atop the wall 118. The substrate 119 was positionedbetween the first chamber 109 and the second chamber 111 of thebiological sterilization indicator 100. The substrate 119 included anaperture 121 formed therethrough of about 3.2 mm (0.125 inch) indiameter, the hole was approximately centered in the substrate. Thesubstrate 119 was positioned between (e.g., sandwiched between) theinsert 130 and the wall 118. The substrate 119 was formed of a chargednylon, and particularly, was a reprobing, charged transfer membraneavailable from GE Water & Process Technologies, Trevose, Pa., under thetrade designation “MAGNAPROBE” (0.45 micron pore size, 30 cm×3 m roll,Catalog No. NP0HY00010, Material No. 1226566).

The biological sterilization indicator 100 had a vent feature 162 asshown in FIG. 7, positioned to fluidly couple the second chamber 111with the first chamber 109. Also, as shown in FIG. 7, the biologicalsterilization indicator 100 had a rib or protrusion 165 that wasintegrally formed with a wall 108 of the housing 102, which waspositioned to maintain the spore carrier 135 in a desired location inthe housing 102.

The housing 102 was tapered (see, e.g., the tapered portion 146 in FIG.6) so that the cross-sectional area in the housing 102 generallydecreased along the longitudinal direction D_(L).

Example 1 Correlation of Fluorescence Readings with Growth after 24Hours

Biological indicators (BI) of the design shown in FIGS. 4-7 anddescribed above in Preparatory Example 1 were built with ˜1×10⁷ CFU of aG. stearothermophilus ATCC 7953 spore crop. Some of the BI's (resultsshown in Tables 26 and 27 and discussed below) were made without thesubstrate material. The liquid growth media 122 was the same as thatused in 3M ATTEST 1292 Rapid Readout Biological Indicators for SteamSterilizers, available from 3M Company of St. Paul. Each BI was then runthrough a steam sterilization cycle of varying lengths of 1 minute, 1minute 45 seconds, 2 minutes, 2 minutes 15 seconds, 2 minutes 30seconds, and 3 minutes at 270° F./132° C. Gravity Steam in a H&W SteamResistometer (available from H&W Technology LLC, Rochester, N.Y.).Following sterilization, the BI's were allowed to cool and activated ina 490 AUTOREADER reading apparatus, available from 3M Company, St. Paul,Minn., similar to the 290 AUTOREADER reading apparatus, available from3M Company; certain features of the 490 AUTOREADER reading apparatus aredescribed in International Publication Nos. WO 2012/061229 and WO2012/061228. Fluorescent readings at excitation/emission 365/460 nm weretaken every 1 minute for 60 minutes. If fluorescence was detected, itwas reported as “YES;” if no fluorescence was detected, it was reportedas “NF” (i.e., no fluorescence). Also, after 24 hours of incubation inthe reading apparatus, the BIs were removed and evaluated for growth,based on a color change (of the pH indicator) in the media from purpleto yellow. If the color change was observed, it was reported as “YES;”if no color change was observed, it was reported as “NO.”

The results shown in Table 24 and Table 25, below, indicate a goodcorrelation between the fluorescence results and the 24 hour growthconfirmation results when the substrate is positioned in the firstlocation for all the BIs exposed to all lengths of sterilization cycles.

The results shown in Table 26 and Table 27, below, indicate inconsistentresults were observed for the BIs when no substrate was present,particularly at the 2 minutes 15 seconds, 2 minutes 30 seconds and 3minute cycle times.

TABLE 24 Fluorescence observations Cycle Time Fluorescence; n = 5 1:00YES YES YES YES YES 1:45 YES YES YES YES YES 2:00 YES YES YES YES YES2:15 NF YES NF YES YES 2:30 NF NF NF NF NF 3:00 NF NF NF NF NF

TABLE 25 Growth observations (color change, after 24 hrs) Cycle TimeGrowth after 24 hrs; n = 5 1:00 YES YES YES YES YES 1:45 YES YES YES YESYES 2:00 YES YES YES YES YES 2:15 NO YES NO YES YES 2:30 NO NO NO NO NO3:00 NO NO NO NO NO

TABLE 26 No Substrate - Fluorescence Cycle Time Fluorescence; n = 5 1:00YES YES YES YES YES 1:45 YES YES YES YES YES 2:00 YES YES YES YES YES2:15 YES NF NF YES YES 2:30 NF NF NF NF NF 3:00 NF NF NF NF NF

TABLE 27 No Substrate - Growth after 24 hrs Cycle Time Growth after 24hrs; n = 5 1:00 YES YES YES YES YES 1:45 YES YES YES YES YES 2:00 YESYES YES YES YES 2:15 NO NO YES YES YES 2:30 NO NO NO NO NO 3:00 NO NO NONO NO

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The complete disclosures of all patents, patent applications,publications, and nucleic acid and protein database entries which arecited herein, are hereby incorporated by reference as if individuallyincorporated. Various modifications and alterations of this inventionwill become apparent to those skilled in the art without departing fromthe scope and spirit of this invention, and it should be understood thatthis invention is not to be unduly limited to the illustrativeembodiments set forth herein.

The invention claimed is:
 1. A method of detecting a biologicalactivity, comprising: providing a sample comprising a source of one ormore predetermined biological activities; a first indicator systemcomprising a pH indicator with a first absorbance spectrum, wherein thepH indicator can be converted by a first predetermined biologicalactivity to a first biological derivative; a second indicator systemcomprising a fluorogenic reagent that is converted by a predeterminedbiological activity to a fluorescent derivative with a second emissionspectrum; a substrate that receives and concentrates the pH indicatorfrom an aqueous mixture; and an instrument that detects the pH indicatorand/or the fluorescent derivative, wherein the instrument comprises anoptical path; forming a first aqueous mixture comprising the sample, thepH indicator, and the fluorogenic reagent; bringing the first aqueousmixture into fluid communication with the substrate to form a secondaqueous mixture; and detecting a presence or absence of fluorescencefrom the fluorescent derivative, wherein detecting a presence or absenceof fluorescence from the fluorescent derivative comprises using theinstrument to detect the fluorescent derivative, wherein the opticalpath does not intersect any portion of the substrate; and observing thesubstrate to detect the pH indicator or the first biological derivative;wherein the first absorbance spectrum includes detectable absorbance inat least a portion of wavelengths present in the second emissionspectrum.
 2. The method of claim 1, wherein, detecting the presence orabsence of fluorescence from the fluorescent derivative comprisesdetecting the presence or absence of fluorescence in the second aqueousmixture.
 3. The method of claim 1, wherein a concentration of pHindicator in the first aqueous mixture is sufficient to preventdetection of an otherwise detectable amount of the fluorescentderivative.
 4. The method of claim 1, further comprising exposing thebiological activity to a sterilant.
 5. The method of claim 1, furthercomprising exposing the biological activity to a temperature shift for aperiod of time.
 6. The method of claim 1, wherein the pH indicatorcomprises a chromophore, wherein detecting the first biologicalderivative comprises detecting a color.
 7. The method of claim 1,wherein detecting the presence or absence of the second biologicalderivative further comprises measuring a quantity of the fluorescentderivative.
 8. The method of claim 1, wherein detecting the presence orabsence of the first biological derivative further comprises measuring aquantity of the first biological derivative.
 9. The method of claim 1,further comprising: providing an instrument that detects the pHindicator and the fluorescent derivative; and using the instrument todetect the pH indicator and the fluorescent derivative.
 10. The methodof claim 9, wherein the housing of the instrument includes: a firstportion, and a second portion adapted to be coupled to the firstportion, the second portion being movable with respect to the firstportion, when coupled to the first portion, between a first position anda second position; wherein the method further comprises moving thesecond portion of the housing from the first position to the secondposition.